US9170249B2 - N-acetylhexosamine-containing N-glycans in glycoprotein products - Google Patents

N-acetylhexosamine-containing N-glycans in glycoprotein products Download PDF

Info

Publication number
US9170249B2
US9170249B2 US13/417,895 US201213417895A US9170249B2 US 9170249 B2 US9170249 B2 US 9170249B2 US 201213417895 A US201213417895 A US 201213417895A US 9170249 B2 US9170249 B2 US 9170249B2
Authority
US
United States
Prior art keywords
glycoprotein
preparation
glycan
glycans
glycoprotein preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
US13/417,895
Other languages
English (en)
Other versions
US20120295273A1 (en
Inventor
Nathaniel J. Washburn
Enrique Arevalo
Kevin Millea
Carlos J. Bosques
Jay Duffner
Brian E. Collins
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Momenta Pharmaceuticals Inc
Original Assignee
Momenta Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=46831284&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US9170249(B2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
US case filed in Delaware District Court litigation https://portal.unifiedpatents.com/litigation/Delaware%20District%20Court/case/1%3A26-cv-00222 Source: District Court Jurisdiction: Delaware District Court "Unified Patents Litigation Data" by Unified Patents is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Momenta Pharmaceuticals Inc filed Critical Momenta Pharmaceuticals Inc
Priority to US13/417,895 priority Critical patent/US9170249B2/en
Assigned to MOMENTA PHARMACEUTICALS, INC. reassignment MOMENTA PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DUFFNER, JAY, MILLEA, KEVIN, WASHBURN, NATHANIEL, BOSQUES, CARLOS J., AREVALO, Enrique, COLLINS, BRIAN EDWARD
Publication of US20120295273A1 publication Critical patent/US20120295273A1/en
Priority to US14/848,768 priority patent/US9890410B2/en
Application granted granted Critical
Publication of US9170249B2 publication Critical patent/US9170249B2/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen

Definitions

  • the present invention relates to methods and materials for the detection of particular glycan structures in glycoproteins.
  • Antibodies represent a growing class of biotherapeutic drugs in the pharmaceutical market and in development for a variety of indications. Unlike small molecules, biologics are actually a mixture of isoforms all of which are contain the same peptide backbone, but differ in modifications and which may have a range of pharmacokinetic, pharmacodymanic or safety profiles. It is therefore important to routinely monitor product quality. Antibodies are glycoproteins which contain at least one variably occupied N-glycosylation site. N-glycosylation in antibody therapeutics is thought to influence pharmacokinetics and structural integrity of the molecule (Krapp, Mimura et al. 2003).
  • the present invention is based, in part, on the discovery of an N-acetylhexosamine glycan structure that can be present on glycoproteins, e.g., a glycosylated antibody.
  • the presence or amount of the N-acetylhexosamine is associated with a particular production parameter or parameters.
  • the absence, presence and/or amount of this glycan structure can be used, inter alia, for evaluating or processing a glycoprotein preparation, e.g., to determine whether to accept or reject a batch of a glycoprotein, e.g., a glycosylated antibody, or to guide or control a production parameter or parameters used to produce a glycoprotein, e.g., a glycosylated antibody.
  • a glycoprotein preparation e.g., to determine whether to accept or reject a batch of a glycoprotein, e.g., a glycosylated antibody, or to guide or control a production parameter or parameters used to produce a glycoprotein, e.g., a glycosylated antibody.
  • the absence, presence or amount of this structure in a glycoprotein composition can be compared, e.g., to a reference standard, e.g., to make a decision regarding the glycoprotein preparation, e.g., a decision to classify, select, accept or discard, release or withhold, process into a drug product, ship, move to a different location, formulate, label, package, release into commerce, or sell or offer for sale the glycoprotein, e.g., a glycosylated antibody.
  • the decision can be to accept, modify or reject a production parameter or parameters used to make the glycoprotein, e.g., a glycosylated antibody.
  • the disclosure features methods for evaluating a glycoprotein preparation.
  • the method includes:
  • the determining step may include one or more of the following: (a) isolating a glycoprotein produced by a cell and determining if an N-acetylhexosamine glycan is present on the glycoprotein, (b) isolating a glycoprotein preparation produced by a cell and determining the presence or amount of the N-acetylhexosamine on glycoproteins of the preparation, (c) isolating glycans from a glycoprotein produced by a cell and determining if the glycans of the glycoprotein include an N-acetylhexosamine glycan, and (d) providing at least one peptide from a glycoprotein produced by cell, and determining the presence glycans containing an N-acetylhexosamine on the at least one peptide.
  • the technique used to measure the N-acetylhexosamine can include one or more of the following methods, and combinations of any of these methods: chromatographic methods, mass spectrometry (MS) methods, electrophoretic methods (such as capillary electrophoresis), nuclear magnetic resonance (NMR) methods, monosaccharide analysis, fluorescence methods, UV-VIS absorbance, enzymatic methods, and use of a detection molecule (such as an antibody or lectin).
  • the source of glycans can be selected from the group consisting of: a population of cells, e.g., CHO cells; an isolated glycoprotein, e.g., an isolated glycosylated antibody; peptides derived from the cleavage of a glycoprotein, e.g., a glycosylated antibody; or glycans derived from the glycoprotein.
  • the method includes treating a source of glycans or glycopeptides with one or more enzymes, e.g., PNGase, followed by analysis of the glycan population.
  • this method includes treating the glycopeptides with a chemical to release the glycan, e.g. acid hydrolysis, followed by analysis of the released glycans or monosaccharides and or analysis of the glycan attached to a single amino acid.
  • the method used provides a quantitative measure of an N-acetylhexosamine glycan. In some embodiments, the method used provides a qualitative measure.
  • the method also includes preparing a glycoprotein preparation, cleaving one or more glycans from the glycoprotein preparation (e.g., with one or more enzyme such as PNGase, and determining the absence, presence or amount of an N-acetylhexosamine glycan.
  • the method is conducted during a production run for a therapeutic glycoprotein by obtaining a sample from the cell culture of the production line, e.g., to monitor glycan structure during production.
  • the determining step is repeated at least once over time, e.g., the determining step is repeated at least once, twice, three times or more, during the time period of culture of the cells.
  • the method is conducted during the storage of a glycoprotein by obtaining a sample from the glycoprotein composition, e.g., to monitor glycan structure stability during storage.
  • the determining step is repeated at least once over time, e.g., the determining step is repeated at least once, twice, three times or more, during the storage of the glycoprotein composition.
  • the method is conducted on a glycoprotein composition, e.g., as part of a quality or release testing of the glycoprotein composition.
  • the determining step includes comparing the level of N-acetylhexosamine glycan containing glycoproteins in a first glycoprotein preparation produced from a first population of cells, e.g., produced under a first production parameter or parameters, to the level of N-acetylhexosamine glycan containing glycoprotein in a second glycoprotein preparation produced from a second population of cells and/or the same cells under a different production parameter or parameters.
  • the presence or amount of an N-acetylhexosamine glycan of a glycoprotein preparation is determined and compared to the presence or amount of the N-acetylhexosamine glycan of a glycoprotein produced under a different production parameter or parameters.
  • the method comprise a step of comparing the level of N-acetylhexosamine glycans to a reference standard (e.g., to a control level, or to a range or value in a product specification).
  • a reference standard e.g., to a control level, or to a range or value in a product specification
  • the determining step includes use of a detection molecule which is able to detect the presence or absence of an N-acetylhexosamine glycan.
  • the detection molecule comprises an antibody that is able to bind to N-acetylhexosamine.
  • the detection molecule may comprise a fluorescent moiety, or a radioisotope moiety. In some embodiments this comprises another sugar that forms a covalent linkage to the N-acetylhexosamine.
  • the detection molecule may comprise a fluorescent moiety, or a radioisotope moiety. In some embodiments this comprises another sugar that forms a covalent linkage to the N-acetylhexosamine.
  • the detection molecule may comprise a fluorescent moiety, or a radioisotope moiety.
  • the glycoprotein e.g., the glycosylated antibody
  • a clonal cell population e.g., a clonal CHO cell population.
  • the cell population may be in culture, e.g., or a sample from a cell culture in a bioreactor for manufacturing the glycoprotein, e.g., the glycosylated antibody.
  • the cell population will have been transformed with at least one vector encoding a glycoprotein.
  • the therapeutic glycoprotein may be of human, non-human or synthetic origins.
  • the glycoprotein may be for treatment of humans or veterinary indications.
  • the method further includes a step of evaluating a biological activity of the glycoprotein produced by the cell, e.g., evaluating the presence or level of immunogenic potential of the glycoprotein, e.g., in vitro or in vivo, e.g., in an animal model.
  • the invention comprises methods for screening one or more cells for the ability to produce an N-acetylhexosamine glycan on a glycoprotein, the method comprising:
  • the cell population is a CHO cell population.
  • the N-acetylhexosamine glycans can be obtained and measured from glycoproteins produced by the cell preparations, from an isolated glycoprotein of the cell preparations, from peptides obtained from a glycoprotein produced by the cell preparations, or from glycan preparations obtained from the cell preparations or from a glycoprotein product thereof.
  • the screening method further comprises the step of isolating a glycoprotein expression product from the cell culture and measuring for the presence or amount of an N-acetylhexosamine glycan on a glycoprotein produced by the cells in step (c).
  • the cell screening method comprises quantifying the amount of N-acetylhexosamine glycans present on the glycoprotein preparation.
  • step (b) of the cell screening method takes place in a bioreactor.
  • Each of the plurality of cell populations may comprise a different strain population, a different clonal cell population, or different samples (e.g., samples taken over time) from a cell culture used to manufacture a glycoprotein.
  • the cell population is transformed with at least one vector encoding a glycoprotein, e.g., a glycosylated antibody, e.g., a human or humanized glycosylated antibody.
  • the glycoprotein is a secreted glycoprotein expressed from the cells.
  • the measuring step of the screening method may include any technique disclosed herein for identifying and/or quantifying an N-acetylhexosamine glycan on the glycoprotein.
  • the invention includes a method for evaluating a glycoprotein preparation.
  • the method includes measuring the amount of an N-acetylhexosamine glycan (e.g., an N-acetylglucosamine and/or N-acetylgalactosamine glycan) in a glycoprotein preparation, e.g., a glycosylated antibody preparation.
  • an N-acetylhexosamine glycan e.g., an N-acetylglucosamine and/or N-acetylgalactosamine glycan
  • the glycoprotein preparation is produced in a host cell, e.g., a prokaryotic or eukaryotic host cell.
  • the eukaryotic cell can be, e.g., a yeast, an insect, a fungi, a plant or an animal cell (e.g., a mammalian cell). Exemplary host cells are described herein.
  • the method includes recording the absence, presence or amount of N-aceytlhexosamine glycans in the glycoprotein preparation in a print or computer-readable medium.
  • the method also includes comparing the measured level of N-acetylhexosamine glycan present in the glycoprotein preparation with a reference standard, such as a control or reference specification.
  • a reference standard can be a specification (e.g., an FDA label or Physician's Insert) or quality criterion for a pharmaceutical preparation containing the glycoprotein preparation.
  • the level of N-acetylhexosamine glycans present in a glycoprotein preparation can be measured as the level of N-acetylhexosamine glycans relative to total amount of glycans in a sample, such as a glycoprotein preparation.
  • the technique used to measure N-acetylhexosamine glycan content includes a chromatographic method.
  • the technique used to measure N-acetylhexosamine glycan content includes mass spectrometry (MS) methods.
  • the technique used to measure N-acetylhexosamine glycan content includes electrophoretic methods (such as capillary electrophoresis).
  • the technique used to measure N-acetylhexosamine glycan content includes nuclear magnetic resonance (NMR) methods.
  • the technique used to measure N-acetylhexosamine glycan content includes monosaccharide analysis.
  • the technique used to measure N-acetylhexosamine glycan content includes fluorescence methods.
  • the technique used to measure N-acetylhexosamine glycan content includes UV-VIS absorbance.
  • the technique used to measure N-acetylhexosamine glycan content includes enzymatic methods.
  • the technique used to measure N-acetylhexosamine glycan content includes and use of a detection molecule (such as an antibody).
  • the invention includes a recombinant glycoprotein that has a different level of N-acetylhexosamine glycans than a reference glycoprotein that has the same or highly similar amino acid sequence.
  • a highly similar amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.
  • the reference glycoprotein is a commercially available therapeutic glycoprotein, e.g., a therapeutic glycoprotein disclosed in Table 2.
  • the recombinant glycoprotein can have a higher or lower level of N-acetylhexosamine glycans than the reference glycoprotein, e.g., the recombinant glycoprotein can have at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% higher or lower level of N-acetylhexosamine glycans, e.g., as measured as a percent of total glycans, or as measured relative to the total amount of G0F, G1F, G2F and glycosylated glycopeptides.
  • the recombinant glycoprotein is an IgG1 preparation, and the recombinant glycoprotein has a level of N-acetylhexosamine glycans greater than or less than 3%, 5%, 10%, or 15% as measured relative to the total amount of G0F, G1F, G2F and glycosylated glycopeptides.
  • the recombinant glycoprotein is an IgG1 preparation, and the recombinant glycoprotein has a level of N-acetylhexosamine glycans greater than or less than 40%, 50% or 55% as measured relative to the total amount of G0F, G1F, G2F and glycosylated glycopeptides.
  • the recombinant glycoprotein is an IgG2 preparation, and the recombinant glycoprotein has a level of N-acetylhexosamine glycans greater than or less than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, or 25% as measured relative to the total amount of G0F, G1F, G2F and glycosylated glycopeptides.
  • the disclosure features a method for modulating effector function of an antibody preparation.
  • the method includes:
  • the method comprises increasing N-acetylhexosamine glycan content of the antibody preparation, e.g., to thereby decrease effector function of the antibody preparation.
  • the method comprises removing one or more glycan structure associated with effector function (e.g., a sialylated glycan), and e.g., the addition of an N-acetylhexosamine glycan.
  • the method comprises enzymatically or chemically modifying the glycan structure to form an N-acetylhexosamine glycan, e.g., with enzymes or chemicals described herein.
  • the method comprises decreasing N-acetylhexosamine glycan content of the antibody preparation, e.g., to thereby increase effector function of the antibody preparation.
  • the method comprises removing one or more N-acetylhexosamine glycans, and e.g., the addition of glycan structure associated with effector function, e.g., a sialylated glycan.
  • the method comprises enzymatically or chemically modifying the glycan structure to form a glycan structure associated with increased effector function.
  • the disclosure features a method for evaluating effector function of an antibody preparation.
  • the method includes:
  • FIG. 1 is a representation of the N-acetylhexosamine glycan structures.
  • FIG. 2 depicts MS/MS fragmentation of IgG Fc glycopeptides containing a single N-linked N-acetylhexosamine glycan.
  • the fragmentation pattern places the modification in the Asn residue, residue 297, in the middle of the sequence (SEQ ID NO: 1).
  • FIG. 3 is a graph depicting the percentage of truncated N-linked N-acetylhexosamine glycan present in commercially available IgG glycoproteins. Relative abundance of the N-acetylhexosamine glycan containing glycoproteins is calculated based upon the abundance of the G0F, G1F, G2F and the aglycosyl glycopeptides. These species comprise more than 85% of the glycan composition for each of the commercially available IgGs tested.
  • detecting As used herein, the terms “detecting,” “detection” and “detecting means” are used interchangeably to refer to the determination of whether a particular chemical moiety, such as an N-acetylhexosamine (e.g., an N-acetylglucosamine and/or N-acetylgalactosamine), is present or absent in or on a compound, preparation, composition, cell or cell population.
  • the detecting means may involve a selectable marker, or an identifiable characteristic such as a fluorescent or radioactive moiety, and may involve labeling of a reagent, compound, cell or cell population.
  • Detection can also refer to the analysis of a compound, preparation, composition, cell or cell population, using such techniques as mass spectrometry or related methods, electrophoretic methods, nuclear magnetic resonance, chromatographic methods, or combinations of the above, to determine the presence or absence of a chemical moiety in or on a compound, preparation, composition, cell or cell population. Detection may also involve quantification of the absolute or relevant levels of the chemical moiety being detected.
  • Glycans are sugars. Glycans can be monomers or polymers of sugar residues, and can be linear or branched. A glycan may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose, phosphomannose, 6′sulfo N-acetylglucosamine, etc.).
  • natural sugar residues e.g., glucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose,
  • glycocan includes homo and heteropolymers of sugar residues.
  • glycan also encompasses a glycan component of a glycoprotein (e.g., of a glycoprotein, glycolipid, proteoglycan, etc.).
  • the term also encompasses free glycans, including glycans that have been cleaved or otherwise released from a glycoprotein.
  • Glycan Preparation refers to a set of glycans obtained according to a particular production method. In some embodiments, glycan preparation refers to a set of glycans obtained from a glycoprotein preparation (see definition of glycoprotein preparation below). In some embodiments, a glycan preparation includes glycoproteins. In some embodiments, a glycan preparation includes released glycans.
  • Glycoprotein refers to a “protein” (as defined herein) that contains a peptide backbone covalently linked to one or more sugar moieties (i.e., glycans).
  • the peptide backbone typically comprises a linear chain of amino acid residues.
  • the sugar moiety(ies) may be in the form of monosaccharides, disaccharides, oligosaccharides, and/or polysaccharides.
  • the sugar moiety(ies) may comprise a single unbranched chain of sugar residues or may comprise one or more branched chains.
  • sugar moieties may include sulfate and/or phosphate groups.
  • sugar moieties may include acetyl, glycolyl, propyl or other alkyl modifications.
  • glycoproteins contain O-linked sugar moieties; in certain embodiments, glycoproteins contain N-linked sugar moieties.
  • Individual molecules of a particular glycoprotein within a glycoprotein preparation typically have identical amino acid sequences but may differ in the occupancy of the at least one glycosylation sites and/or in the identity of the glycans linked to the at least one glycosylation sites. That is, a glycoprotein preparation may contain only a single glycoform of a particular glycoprotein, but more typically contains a plurality of glycoforms. Different preparations of the same glycoprotein may differ in the identity of glycoforms present (e.g., a glycoform that is present in one preparation may be absent from another) and/or in the relative amounts of different glycoforms.
  • Glycoprotein Composition refers to a glycoprotein preparation that is in the form of a drug substance or drug product.
  • glycosidase refers to an agent that cleaves a covalent bond between sequential sugars in a glycan or between the sugar and the backbone moiety (e.g., between sugar and peptide backbone of glycoprotein).
  • a glycosidase is an enzyme.
  • a glycosidase is a protein (e.g., a protein enzyme) comprising one or more polypeptide chains.
  • a glycosidase is a chemical cleavage agent, e.g., hydrazine.
  • N-glycan refers to a polymer of sugars that has been released from a glycoprotein but was formerly linked to a glycoprotein via a nitrogen linkage (see definition of N-linked glycan below).
  • N-linked Glycans are glycans that are linked to a glycoprotein via a nitrogen linkage.
  • a diverse assortment of N-linked glycans exists, but is typically based on the common core pentasaccharide (Man) 3 (GlcNAc)(GlcNAc).
  • O-glycan refers to a polymer of sugars that has been released from a glycoconjugate but was formerly linked to the glycoconjugate via an oxygen linkage (see definition of O-linked glycan below).
  • O-linked Glycans are glycans that are linked to a glycoconjugate via an oxygen linkage.
  • O-linked glycans are typically attached to glycoproteins via N-acetyl-D-galactosamine (GalNAc) or via N-acetyl-D-glucosamine (GlcNAc) to the hydroxyl group of L-serine (Ser) or L-threonine (Thr).
  • GalNAc N-acetyl-D-galactosamine
  • GlcNAc N-acetyl-D-glucosamine
  • Some O-linked glycans also have modifications such as acetylation and sulfation.
  • Modulate refers to the ability of an actor to control, within prescribed limits, the value of a parameter, such as the level of N-acetylhexosamine glycans present in a glycoprotein preparation.
  • a parameter such as the level of N-acetylhexosamine glycans present in a glycoprotein preparation.
  • the level of N-acetylhexosamine glycans may be modulated so that it remains within prescribed limits.
  • the level of N-acetylhexosamine glycans may be modulated so that it does not vary by more than 10.0%, 5.0%, 1.0%, 0.5% or 0.1% of a reference standard.
  • protease refers to an agent that cleaves a peptide bond between sequential amino acids in a polypeptide chain.
  • a protease is an enzyme (i.e., a proteolytic enzyme).
  • a protease is a protein (e.g., a protein enzyme) comprising one or more polypeptide chains.
  • a protease is a chemical cleavage agent.
  • the term “providing” as used herein refers to an actor obtaining a subject item, such as a cell preparation, or glycoprotein preparation, from any source including, but not limited to, obtaining by the actor's own manufacture or by the actor's receiving the item from another party.
  • a cell preparation is provided if it is made or received by any machine, person, or entity.
  • a cell preparation may be received by a machine, which may then perform one or more tests, processes, or refinements of the glycoprotein preparation.
  • a cell preparation may be received by a person.
  • a CHO cell preparation may be received from an outside entity.
  • a cell preparation may be received by a person or business performing characterization services for a second person or business.
  • N-acetylhexosamine Glycan The term “N-acetylhexosamine glycan” as used herein, describes the glycan structures illustrated in FIG. 1 .
  • glycoproteins Although host cells used for the synthesis of recombinant glycoproteins possess the intracellular machinery to produce complex glycosylation, these cells do not always possess the same complement of enzymes as the cells in which the glycoprotein is naturally expressed. Clonal selection of cell lines and variations in manufacturing conditions may also produce heterogeneity in glycoproteins expressed in cultured cells. The functional role of glycosylation in glycoprotein activity necessitates careful characterization of therapeutic products produced in cell lines.
  • glycoprotein preparations e.g., glycosylated antibody preparations
  • glycoprotein preparations e.g., glycosylated antibody preparations
  • an unusual N-linked N-acetylhexosamine glycan structure For example, it has been found that an N-linked N-acetylhexosamine glycan can be found at the glycosylation site Asn297 of the Fc region of antibodies within a glycosylated antibody preparation. Many glycosylated antibody preparations contain this structure and, thus it is important to identify, monitor and control this aspect of glycan structure when producing glycosylated antibody preparations.
  • the present disclosure provides methods of analyzing the composition of glycans on glycoproteins.
  • glycans from glycoprotein preparations can be analyzed to determine whether they include an N-acetylhexosamine glycan.
  • the present disclosure provides methods of detecting the absence, presence or amount of N-acetylhexosamine glycans associated with a glycoprotein preparation, e.g., a glycosylated antibody preparation, e.g., a glycosylated antibody preparation described herein, and methods of producing glycoproteins that include, include is a certain amount or lack this glycan structure.
  • the present disclosure provides methods of analyzing the structure and/or composition of individual glycans within a glycan preparation, e.g., evaluating for the absence, presence or amount of N-acetylhexosamine glycans (N-acetylglucosamine and/or N-acetylgalactosamine).
  • a glycan preparation may be obtained from a cell preparation or from a glycoprotein made by any method available in the art.
  • obtaining a glycan preparation comprises steps of (1) obtaining a cell or glycoprotein preparation; and (2) optionally releasing glycans from the cell or glycoprotein preparation.
  • obtaining a glycan preparation optionally comprises labeling the glycan preparation with a detectable label.
  • glycoproteins secreted by cultured cells can be isolated and purified by any available means, such as anion-exchange chromatography, reversed-phase chromatography, gel filtration, immunoaffinity chromatography, and combinations thereof.
  • an N-glycan preparation is obtained by providing a glycoprotein population and removing N-linked glycans from the glycoproteins in the population.
  • N-linked glycans are removed from glycoproteins (e.g., glycoproteins) by digestion.
  • glycoproteins e.g., glycoproteins
  • glycanases to be used in accordance with the present disclosure cleave between GlcNAc-Asn, GlcNAc-GlcNAc, or Man-GlcNAc residues of the core.
  • Exemplary enzymes which can be used to remove N-linked glycans from glycoproteins include, but are not limited to, N-glycanase F and/or N-glycanase-A, O-glycanase and/or Endo H.
  • N-linked glycans are removed from glycoproteins by chemical cleavage.
  • hydrazine, sodium borohydride, and/or trifluoromethanesulfonic acid (TFMS) can be used to remove glycans from a glycoprotein.
  • an O-linked glycan preparation is obtained by providing a glycoprotein (e.g., glycoprotein) population and removing O-linked glycans from glycoproteins in the population.
  • a glycoprotein e.g., glycoprotein
  • O-linked glycans are removed from glycoproteins (e.g., glycoproteins) by beta elimination. In some embodiments, O-linked glycans are removed from glycoproteins (e.g., glycoproteins) by reductive beta elimination. In some embodiments, O-glycans are removed from glycoproteins (e.g., glycoproteins) by non-reductive beta elimination.
  • O-linked glycans are removed from a glycoprotein (e.g., a glycoprotein) preparation by incubating the preparation in a solution that includes alkaline tetrahydroborate.
  • tetradeuterioborate is used, e.g., to incorporate a deuterium label to facilitate detection of O-linked glycans.
  • a glycoprotein preparation is incubated in a solution containing 0.8-1.0 M NaBH 4 and 0.05-0.1 M NaOH at 42-45° C. for 2-24 hours.
  • a reaction to remove O-linked glycans can be terminated by the addition of acid (e.g., 1.0 M HCl).
  • O-linked glycans are removed from a glycoprotein preparation by incubating the preparation in a solution that includes NaOH.
  • a glycoprotein is incubated in a solution containing 50-200 mM NaOH at 27-45° C. for 2-48 hours. A reaction can be terminated by the addition of acid.
  • O-linked glycans are removed from a glycoprotein preparation by incubating the preparation in a solution that includes NH 4 OH.
  • a glycoprotein is incubated in a solution containing 25-28% NH 4 OH at 45-60° C. for 2-40 hours. The reaction can be terminated by removing the NH 4 OH under vacuum.
  • the solution includes ammonium carbonate (e.g., at a saturating concentration).
  • the NH 4 OH-treated preparation is treated with acid (e.g., boric acid).
  • O-linked glycans are removed from a glycoprotein preparation by incubating the preparation in an aqueous solution that includes ethylamine (e.g., ethylamine at about 70%) or methylamine (e.g., methylamine at about 40%), for about 4-24 hours.
  • ethylamine e.g., ethylamine at about 70%
  • methylamine e.g., methylamine at about 40%
  • an O-linked glycan preparation is obtained from a glycoprotein population from which N-linked glycans have been removed.
  • labels can be associated with glycans before or after release from a glycoprotein.
  • N-linked glycans or O-linked glycans e.g., N-glycans that have been removed from a glycoprotein population
  • Detectable labels are typically associated with the reducing ends of glycans.
  • detectable labels are fluorescent moieties.
  • Exemplary fluorophores that can be used in accordance with the present disclosure include, but are not limited to, 2-aminobenzoic acid (2AA), 2-aminobenzamide (2AB), and/or 2-aminopurine (2AP).
  • fluorophores for use in accordance with the present disclosure are characterized by having reactivity with the reducing end of an oligosaccharide and/or monosaccharide under conditions that do not damage and/or destroy the glycan.
  • fluorescent moieties are attached to reducing ends directly.
  • direct attachment can be accomplished by direct conjugation by reductive amination.
  • fluorescent moieties are attached to reducing ends indirectly.
  • indirect attachment can be accomplished by a reactive linker arm.
  • detectable labels comprise radioactive moieties or isotopically-labelled molecules.
  • radioactive moieties that can be used in accordance with the present disclosure include, but are not limited to, tritium ( 3 H), deuterium ( 2 H), and/or 35 S. Typically, such moieties are directly attached to or otherwise associated with the fluorophore.
  • 2AP can be modified such that all hydrogens are deuterated.
  • the present disclosure provides improved methods of determining glycosylation patterns of glycoproteins. Such methods can involve subjecting a glycan population to one or more exoglycosidases and analyzing the structure and/or composition of the digestion products.
  • exoglycosidases used in accordance with the present disclosure recognize and cleave only one particular type of glycosidic linkage.
  • exoglycosidases used in accordance with the present disclosure recognize and cleave more than one particular type of glycosidic linkage.
  • exoglycosidases which may be useful for the present invention are ⁇ -galactosidases, ⁇ -galactosidases; hexosaminidases, mannosidases; and combinations thereof.
  • Exoglycosidases are enzymes which cleave terminal glycosidic bonds from the non-reducing end of glycans. They are typically highly specific to particular monosaccharide linkages and anomericity ( ⁇ / ⁇ ). In some embodiments, neighboring branching patterns can affect exoglycosidase specificity. Exoglycosidase treatment usually results in glycans of standard antennary linkages being cleaved down to the pentasaccharide core (M3N2) containing 3 mannose and 2 GlcNAc residues.
  • M3N2 pentasaccharide core
  • unusually-modified species e.g., antennary or core fucosylated species, high-mannose and hybrid glycans, lactosamine-extended glycans, sulfated glycans, phosphorylated glycans, etc.
  • antennary or core fucosylated species e.g., high-mannose and hybrid glycans, lactosamine-extended glycans, sulfated glycans, phosphorylated glycans, etc.
  • exoglycosidases that can be used in accordance with the present disclosure include, but are not limited to, sialidase, galactosidase, hexosaminidase, fucosidase, and mannosidase.
  • Exoglycosidases can be obtained from any source, including commercial sources or by isolation and/or purification from a cellular source (e.g., bacteria, yeast, plant, etc.).
  • exoglycosidases e.g., sialidases, galactosidases, hexosaminidases, fucosidases, and mannosidases
  • exoglycosidases can be divided into multiple categories or “subsets.” In some embodiments, the different subsets display different abilities to cleave different types of linkages.
  • Table 1 presents some exemplary exoglycosidases, their linkage specificities, and the organism from which each is derived.
  • this is an exemplary, not a comprehensive, list of exoglycosidases, and that any exoglycosidase having any linkage specificity may be used in accordance with the present disclosure.
  • a glycan population can be digested with any exoglycosidase or any set of exoglycosidases.
  • exoglycosidase reactions take place under conditions that are compatible with enzyme activity. For example, pH, temperature, reaction solution components and concentration (e.g., salt, detergent, etc.), and length of reaction time can be optimized in order to achieve a desired level of exoglycosidase activity. See, e.g., WO 2008/130926, the contents of which are herein incorporated by reference.
  • methods in accordance with the disclosure comprise subjecting a glycan preparation to analysis to determine whether glycoproteins in the preparation include an N-acetylhexosamine glycan structure.
  • the analysis comprises comparing the structure and/or function of glycans in one glycoprotein preparation from one source to structure and/or function of glycans in at least one other glycoprotein preparation from another source.
  • the analysis comprises comparing the structure and/or function of glycans in one or more of the samples to structure and/or function of glycans in a reference sample.
  • glycan structure and composition can be analyzed by any available method.
  • glycan structure and composition are analyzed by chromatographic methods, mass spectrometry (MS) methods, chromatographic methods followed by MS, electrophoretic methods, electrophoretic methods followed by MS, nuclear magnetic resonance (NMR) methods, and combinations thereof.
  • MS mass spectrometry
  • NMR nuclear magnetic resonance
  • glycan structure and composition can be analyzed by chromatographic methods, including but not limited to, liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof.
  • LC liquid chromatography
  • HPLC high performance liquid chromatography
  • UPLC ultra performance liquid chromatography
  • TLC thin layer chromatography
  • amide column chromatography amide column chromatography
  • glycan structure and composition can be analyzed by mass spectrometry (MS) and related methods, including but not limited to, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof.
  • MS mass spectrometry
  • MS mass spectrometry
  • MALDI-MS matrix assisted laser desorption ionisation mass spectrometry
  • FTMS Fourier transform mass spectrometry
  • IMS-MS ion mobility separation with mass spectrometry
  • ETD-MS electron transfer dissociation
  • glycan structure and composition can be analyzed by electrophoretic methods, including but not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, and combinations thereof.
  • electrophoretic methods including but not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, and combinations thereof.
  • glycan structure and composition can be analyzed by nuclear magnetic resonance (NMR) and related methods, including but not limited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), and combinations thereof.
  • NMR nuclear magnetic resonance
  • glycans are analyzed in accordance with the present disclosure using one or more available methods (to give but a few examples, see Anumula, Anal. Biochem. 350 (1):1, 2006; Hara et al., Anal. Biochem., 179:162, 1989; and/or Townsend, R. R. Carbohydrate Analysis” High Performance Liquid Chromatography and Capillary Electrophoresis., Ed. Z. El Rassi, pp 181-209, 1995, each of which is incorporated herein by reference in its entirety).
  • glycans are characterized using one or more of chromatographic methods, electrophoretic methods, nuclear magnetic resonance methods, and combinations thereof.
  • Exemplary such methods include, for example, NMR, mass spectrometry, liquid chromatography, 2-dimensional chromatography, SDS-PAGE, antibody staining, lectin staining, monosaccharide quantitation, capillary electrophoresis, fluorophore-assisted carbohydrate electrophoresis (FACE), micellar electrokinetic chromatography (MEKC), exoglycosidase or endoglycosidase treatments, and combinations thereof.
  • FACE fluorophore-assisted carbohydrate electrophoresis
  • MEKC micellar electrokinetic chromatography
  • exoglycosidase or endoglycosidase treatments and combinations thereof.
  • methods described herein allow for detection of glycan species (such as an N-acetylhexosamine glycan (e.g., an N-acetylglucosamine and/or N-acetylgalactosamine glycan) that are present at low levels within a population of glycans.
  • glycan species such as an N-acetylhexosamine glycan (e.g., an N-acetylglucosamine and/or N-acetylgalactosamine glycan) that are present at low levels within a population of glycans.
  • the present methods allow for detection of glycan species that are present at levels less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.1%, less than 0.075%, less than 0.05%, less than 0.025%, or less than 0.01% within a population of glycans.
  • methods described herein allow for detection of particular structures (e.g., an N-acetylhexosamine glycan (e.g., an N-acetylglucosamine and/or N-acetylgalactosamine glycan)) that are present at low levels within a population of glycans.
  • particular structures e.g., an N-acetylhexosamine glycan (e.g., an N-acetylglucosamine and/or N-acetylgalactosamine glycan)
  • the present methods allow for detection of particular structures that are present at levels less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.1%, less than 0.075%, less than 0.05%, less than 0.025%, or less than 0.01% within a population of glycans.
  • methods described herein allow for detection of relative levels of individual glycan species within a population of glycans. For example, the area under each peak of a liquid chromatograph can be measured and expressed as a percentage of the total. Such an analysis provides a relative percent amount of each glycan species within a population of glycans.
  • relative levels of individual glycan species are determined from areas of peaks in a 1D-NMR experiment, or from volumes of cross peaks from a 1H-15HSQC spectrum (e.g., with correction based on responses from standards), or by relative quantitation by comparing the same peak across samples.
  • a biological activity of a glycoprotein preparation is assessed.
  • Biological activity of the glycoprotein preparation can be analyzed by any available method.
  • a binding activity of a glycoprotein is assessed (e.g., binding to a receptor).
  • a therapeutic activity of a glycoprotein is assessed (e.g., an activity of a glycoprotein in decreasing severity or symptom of a disease or condition, or in delaying appearance of a symptom of a disease or condition).
  • a pharmacologic activity of a glycoprotein is assessed (e.g., bioavailability, pharmacokinetics, pharmacodynamics).
  • glycoprotein preparations can be analyzed by any available method.
  • immunogenicity of a glycoprotein preparation is assessed, e.g., by determining whether the preparation elicits an antibody response in a subject.
  • biological activity, therapeutic activity, etc., of a glycoprotein preparation that includes an N-acetylhexosamine glycan is compared to a glycoprotein preparation that does not include or includes at background levels an N-acetylhexosamine glycan.
  • biological activity, therapeutic activity, etc., of a glycoprotein preparation having N-acetylhexosamine glycans is compared to a glycoprotein preparation having a different level of N-acetylhexosamine glycans.
  • Methods of the present disclosure can be utilized to analyze glycans from glycoproteins in any of a variety of states including, for instance, free glycans, glycoproteins (e.g., glycopeptides, glycolipids, proteoglycans, etc.), cell-associated glycans (e.g., nucleus-, cytoplasm-, cell-membrane-associated glycans, etc.); glycans associated with cellular, extracellular, intracellular, and/or subcellular components (e.g., proteins); glycans in extracellular space (e.g., cell culture medium), etc.
  • glycoproteins e.g., glycopeptides, glycolipids, proteoglycans, etc.
  • cell-associated glycans e.g., nucleus-, cytoplasm-, cell-membrane-associated glycans, etc.
  • Methods of the present disclosure may be used in one or more stages of process development for the production of a therapeutic or other commercially relevant glycoprotein.
  • the methods described herein can be used to evaluate a production parameter or parameters using to produce a glycoprotein preparation, to compare glycoprotein preparations produced by different production parameters, and to determine and/or select a production parameter or parameters for a glycoprotein preparation such that a particular glycan structure can be obtained upon production of a glycoprotein preparation.
  • a production parameter as used herein is a parameter or element in a production process.
  • Production parameters that can be selected include, e.g., the cell or cell line used to produce the glycoprotein preparation, the culture medium, culture process or bioreactor variables (e.g., batch, fed-batch, or perfusion), purification process and formulation of a glycoprotein preparation.
  • Exemplary production parameters include: 1) the types of host; 2) genetics of the host; 3) media type; 4) fermentation platform; 5) purification steps; and 6) formulation.
  • the present disclosure can also be utilized to monitor the extent and/or type of glycosylation occurring in a particular cell culture (e.g., the extent of N-acetylhexosamine glycans in glycoprotein preparation produced in the cell culture), thereby allowing adjustment or possibly termination of the culture in order, for example, to achieve a particular desired glycosylation pattern or to avoid development of a particular undesired glycosylation pattern.
  • a particular cell culture e.g., the extent of N-acetylhexosamine glycans in glycoprotein preparation produced in the cell culture
  • the present disclosure can also be utilized to assess glycosylation characteristics of cells or cell lines (e.g., CHO cell lines) that are being considered for production of a particular desired glycoprotein (for example, even before the cells or cell lines have been engineered to produce the glycoprotein, or to produce the glycoprotein at a commercially relevant level).
  • cells or cell lines e.g., CHO cell lines
  • the target glycoprotein is a therapeutic glycoprotein, for example having undergone regulatory review in one or more countries, it will often be desirable to monitor cultures to assess the likelihood that they will generate a product with a glycosylation pattern as close to the established glycosylation pattern of the pharmaceutical product as possible (e.g., having a degree of N-acetylhexosamine glycan content which is close to that of the pharmaceutical product), e.g., whether or not it is being produced by exactly the same route.
  • “close” refers to a glycosylation pattern having at least about a 75%, 80%, 85%, 90%, 95%, 98%, or 99% correlation to the established glycosylation pattern of the pharmaceutical product.
  • samples of the production culture are typically taken at multiple time points and are compared with an established standard or with a control culture in order to assess relative glycosylation.
  • methods for monitoring production of a glycoprotein may comprise steps of (i) during production of a glycoprotein, removing at least first and second glycan-containing samples from the production system; (ii) subjecting each of the first and second glycan-containing samples to an analysis to determine whether a particular modification is present (e.g., an N-acetylhexosamine glycan); and (iii) comparing the products obtained from the first glycan-containing sample with those obtained from the second glycan-containing sample so that differences are determined and therefore progress of glycoprotein production is monitored.
  • the production system comprises CHO cells.
  • the present disclosure may be utilized, for example, to monitor glycosylation at particular stages of development, or under particular growth conditions.
  • methods described herein can be used to characterize, modulate and/or control or compare the quality of therapeutic products.
  • the present methodologies can be used to assess glycosylation in cells producing a therapeutic protein product. Particularly given that glycosylation can often affect the activity, bioavailability, or other characteristics of a therapeutic protein product, methods for assessing cellular glycosylation during production of such a therapeutic protein product are particularly desirable.
  • the present disclosure can facilitate real time analysis of glycosylation in production systems for therapeutic proteins, and hence, modulation of the glycosylation may be achieved.
  • the glycoprotein preparation is a glycosylated antibody preparation.
  • antibody refers to a protein that includes at least one immunoglobulin variable domain (variable region) or immunoglobulin variable domain (variable region) sequence.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH or HV), and a light (L) chain variable region (abbreviated herein as VL or LV).
  • VH or HV heavy chain variable region
  • L light chain variable region
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody encompasses antigen-binding fragments of antibodies as well as complete antibodies.
  • antigen-binding fragment” of a full length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest.
  • An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).
  • Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred.
  • the VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds.
  • the heavy chain constant region includes three immunoglobulin domains, CH1, CH2 and CH3.
  • the light chain constant region includes a CL domain.
  • the variable region of the heavy and light chains contains a binding domain that interacts with an antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the glycosylated antibody has one or more regions that are human or effectively human.
  • one or more of the variable regions can be human or effectively human.
  • one or more of the CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3.
  • Each of the light chain (LC) and/or heavy chain (HC) CDRs can be human.
  • HC CDR3 can be human.
  • One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and/or FR4 of the HC and/or LC.
  • the Fc region can be human. In one embodiment, all the framework regions are human.
  • glycoprotein products include, for example, the glycosylated antibodies provided in Table 2:
  • the disclosure provides methods in which glycans from glycoproteins from different sources or samples are compared with one another.
  • multiple samples from the same source e.g., from the same CHO cell source
  • changes in glycosylation patterns e.g., changes in the presence or extent of N-acetylhexosamine glycans
  • one of the samples is a historical sample or a record of a historical sample.
  • one of the samples is a reference sample.
  • the disclosure provides methods in which glycans from glycoproteins expressed by different cell sources are compared with one another.
  • one or more of the compared cell sources are CHO cells.
  • glycans from different cell culture samples prepared under different production parameters e.g., cell type, culture type (e.g., continuous feed vs. batch feed, etc.), culture conditions (e.g., type of media, presence or concentration of particular component of particular medium(a), osmolarity, pH, temperature, timing or degree of shift in one or more components such as osmolarity, pH, temperature, etc.), culture time, isolation steps, etc.
  • production parameters e.g., cell type, culture type (e.g., continuous feed vs. batch feed, etc.), culture conditions (e.g., type of media, presence or concentration of particular component of particular medium(a), osmolarity, pH, temperature, timing or degree of shift in one or more components such as osmolarity, pH, temperature, etc.), culture time, isolation steps, etc.) but are otherwise identical, are compared, so that effects of the selected production parameter on glycosylation are determined.
  • glycans from different cell culture samples prepared under conditions that differ in a single selected production parameter are compared so that effects of the single selected production parameter on glycosylation patterns (e.g., the absence, presence, or extent of N-acetylhexosamine glycans) are determined.
  • effects of the single selected production parameter on glycosylation patterns e.g., the absence, presence, or extent of N-acetylhexosamine glycans
  • use of techniques as described herein may facilitate determination of the effects of particular production parameters on glycosylation patterns in cells.
  • glycans from different batches of a glycoprotein are compared.
  • the present disclosure facilitates quality control of a glycoprotein preparation.
  • some such embodiments facilitate monitoring of progress of a particular culture producing a glycoprotein (e.g., when samples are removed from the culture at different time points and are analyzed and compared to one another).
  • multiple samples from the same source are obtained over time, so that changes in glycosylation patterns are monitored.
  • glycan-containing samples are removed at about 30 second, about 1 minute, about 2 minute, about 5 minute, about 10 minute, about 30 minute, about 1 hour, about 2 hour, about 3 hour, about 4 hour, about 5 hour, about 10 hour, about 12 hour, or about 18 hour intervals, or at even longer intervals. In some embodiments, glycan-containing samples are removed at irregular intervals. In some embodiments, glycan-containing samples are removed at 5 hour intervals.
  • methods in accordance with the disclosure may be used to monitor the glycosylation pattern of glycoproteins during the course of their production by cells.
  • production of a glycoprotein may involve steps of (1) culturing cells that produce the glycoprotein, (2) obtaining samples at regular or irregular intervals during the culturing, and (3) analyzing the glycosylation pattern of produced glycoprotein(s) in obtained sample(s).
  • such methods may comprise a step of comparing the glycosylation patterns of produced glycoprotein(s) in obtained samples to one another.
  • such methods may comprise a step of comparing glycosylation patterns of produced glycoprotein(s) in obtained sample(s) to the glycosylation pattern of a reference sample.
  • methods in accordance with the disclosure may be used to monitor the glycosylation pattern of glycoproteins over the course of storage.
  • the method can include obtaining samples at regular or irregular intervals during the storage of a glycoprotein preparation, and (3) analyzing the glycosylation pattern of glycoprotein(s) in obtained sample(s).
  • such methods may comprise a step of comparing the glycosylation patterns of the glycoprotein(s) in obtained samples to one another.
  • such methods may comprise a step of comparing glycosylation patterns of the glycoprotein(s) in obtained sample(s) to the glycosylation pattern of a reference sample.
  • features of the glycan analysis can be recorded, for example in a quality control record.
  • a comparison is with a historical record of a prior or standard batch and/or with a reference sample of glycoprotein.
  • glycans from different batches of a particular glycoprotein are compared to one another and/or to a reference sample.
  • batch-to-batch comparison may comprise the steps of (i) providing a first glycan preparation from a first batch of the glycoprotein; (ii) providing a second glycan preparation from a second batch of the glycoprotein; (iii) subjecting each of the first and second glycan preparations to analysis procedure; and (iv) comparing the results of the analysis obtained from the first glycan preparation with the cleavage products obtained from the second preparation so that consistency of the two batches is assessed.
  • glycan preparations can be provided by removing at least one glycan from at least one glycoprotein from a batch and, optionally, isolating removed glycans.
  • glycan preparations may be labeled as described herein (e.g., fluorescently and/or radioactively; e.g., prior to and/or after isolation).
  • the present disclosure facilitates quality control of a glycoprotein preparation.
  • Features of the glycan analysis can be recorded, for example in a quality control record.
  • a comparison is with a historical record of a prior or standard batch of glycoprotein.
  • a comparison is with a reference glycoprotein sample.
  • the present disclosure may be utilized in studies to modify the glycosylation characteristics of a cell, for example to establish a cell line and/or culture conditions with one or more desirable glycosylation characteristics, e.g., a cell line that produces glycoproteins having, having a certain amount or lacking an N-acetylhexosamine glycan. Such a cell line and/or culture conditions can then be utilized, if desired, for production of a particular target glycoprotein for which such glycosylation characteristic(s) is/are expected to be beneficial.
  • the cell is a CHO cell.
  • techniques described herein can be used to detect desirable or undesirable glycans, for example to detect or quantify the presence of one or more contaminants in a glycoprotein product, or to detect or quantify the presence of one or more active or desired species.
  • methods described herein facilitate detection of glycans that are present at very low levels in a source (e.g., a biological sample, glycan preparation, etc.).
  • a source e.g., a biological sample, glycan preparation, etc.
  • the levels of glycans comprising between 0.1% and 5%, e.g., between 0.1% and 2%, e.g., between 0.1% and 1% of a glycan preparation.
  • methods described herein allow for detection of relative levels of individual glycan species within a population of glycans. For example, the area under each peak of a liquid chromatograph can be measured and expressed as a percentage of the total. Such an analysis provides a relative percent amount of each glycan species within a population of glycans.
  • methods described herein allow for the manufacture of a glycoprotein, e.g., a glycoprotein containing an N-acetylhexosamine glycan, e.g., N-acetylglucosamine or N-acetylgalactosamine.
  • a glycoprotein e.g., a glycoprotein containing an N-acetylhexosamine glycan, e.g., N-acetylglucosamine or N-acetylgalactosamine.
  • the manufacture of a glycoprotein may involve steps of (1) culturing cells that produce the glycoprotein, (2) obtaining samples of the glycoprotein (e.g., at regular or irregular intervals during the culturing, and/or at the end of a culturing process) and (3) analyzing the glycosylation pattern of produced glycoprotein(s) in obtained sample(s) for the presence, absence and/or amount of an N-acetylhexosamine glycan, e.g., N-acetylglucosamine or N-acetylgalactosamine.
  • an N-acetylhexosamine glycan e.g., N-acetylglucosamine or N-acetylgalactosamine.
  • such methods may comprise a step of comparing the glycosylation patterns of produced glycoprotein(s) in obtained samples to a reference, e.g., comparing the presence, absence and/or amount of an N-acetylhexosamine glycan, e.g., N-acetylglucosamine or N-acetylgalactosamine in the produced glycoprotein, to a reference, such as a pharmaceutical specification, e.g., a pharmaceutical specification for the produced glycoprotein for the presence, absence and/or amount of an N-acetylhexosamine glycan, e.g., N-acetylglucosamine or N-acetylgalactosamine.
  • the methods may comprise a step of comparing glycosylation patterns of produced glycoprotein(s) in obtained sample(s) to the glycosylation pattern of a reference sample.
  • the method comprises further processing of the glycoprotein, e.g., the further processing can include combining the glycoprotein preparation with a second component, e.g., an excipient or buffer.
  • the further processing can include one or more of: formulating the glycoprotein preparation; processing the glycoprotein n preparation into a drug product; combining the glycoprotein preparation with a second component, e.g., an excipient or buffer; changing the concentration of the glycoprotein in the preparation; lyophilizing the glycoprotein preparation; combining a first and second aliquot of the glycoprotein to provide a third, larger, aliquot; dividing the glycoprotein preparation into smaller aliquots; disposing the glycoprotein preparation into a container, e.g., a gas or liquid tight container; packaging the glycoprotein preparation; associating a container comprising the glycoprotein preparation with a label; shipping or moving the glycoprotein preparation to a different location.
  • a container e.g., a gas or liquid tight container
  • packaging the glycoprotein preparation associating a container
  • the truncated N-linked glycan identified was unexpected based on the biosynthetic glycosylation pathway as it is understood.
  • a glycoprotein proceeds through the standard glycosylation cascade resulting in a number of potential N-linked glycan species, all of which contain at least a core structure containing 2 GlcNAc moieties and 3 mannose residues.
  • This species identified consists of only a single N-acetylhexosamine linked to Asn297 in the Fc domain of the antibody. The species was identified though mass spectrometric analysis of the tryptic digest of a monoclonal antibody.
  • Peptides were derived from each of the commercially available antibody products through the use of enzymatic cleavage (i.e. trypsin). Peptides were reduced and alkylated and analyzed by LC-MS with MS/MS used for peptide sequencing. The amino acid sequence from peptide sequencing was used to determine the identity of the peptide. Glycopeptides were observed as a mass shift relative to the non-glycosylated peptide.
  • the relative abundance of HexNAc-glycopeptide in various commercially available antibody products was calculated based on the abundance of the G0F (fucosylated, but no galactose residues biantennery glycan), G1F (fucosylated and one galactose residue biantennery glycan), G2F (fucosylated and two galactose residues, biantennery glycan), and the aglycosyl glycopeptides.
  • Peptides were derived from the drug substance through the use of enzymatic cleavage (i.e. trypsin). Peptides were reduced and alkylated and analyzed by LC-MS with MS/MS used for peptide sequencing. The amino acid sequence from peptide sequencing was used to determine the identity of the peptide. Glycopeptides were observed as a mass shift relative to the non-glycosylated peptide.
  • FIG. 2 shows the fluorescence chromatogram of a fraction of glycans derived from the monoclonal antibody.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
US13/417,895 2011-03-12 2012-03-12 N-acetylhexosamine-containing N-glycans in glycoprotein products Active US9170249B2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US13/417,895 US9170249B2 (en) 2011-03-12 2012-03-12 N-acetylhexosamine-containing N-glycans in glycoprotein products
US14/848,768 US9890410B2 (en) 2011-03-12 2015-09-09 N-acetylhexosamine-containing N-glycans in glycoprotein products

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161452092P 2011-03-12 2011-03-12
US13/417,895 US9170249B2 (en) 2011-03-12 2012-03-12 N-acetylhexosamine-containing N-glycans in glycoprotein products

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/848,768 Continuation US9890410B2 (en) 2011-03-12 2015-09-09 N-acetylhexosamine-containing N-glycans in glycoprotein products

Publications (2)

Publication Number Publication Date
US20120295273A1 US20120295273A1 (en) 2012-11-22
US9170249B2 true US9170249B2 (en) 2015-10-27

Family

ID=46831284

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/417,895 Active US9170249B2 (en) 2011-03-12 2012-03-12 N-acetylhexosamine-containing N-glycans in glycoprotein products
US14/848,768 Active US9890410B2 (en) 2011-03-12 2015-09-09 N-acetylhexosamine-containing N-glycans in glycoprotein products

Family Applications After (1)

Application Number Title Priority Date Filing Date
US14/848,768 Active US9890410B2 (en) 2011-03-12 2015-09-09 N-acetylhexosamine-containing N-glycans in glycoprotein products

Country Status (4)

Country Link
US (2) US9170249B2 (fr)
EP (1) EP2686671A4 (fr)
CN (1) CN103782168B (fr)
WO (1) WO2012125553A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160060675A1 (en) * 2011-03-12 2016-03-03 Momenta Pharmaceuticals, Inc. N-acetylhexosamine-containing n-glycans in glycoprotein products
US9921210B2 (en) 2010-04-07 2018-03-20 Momenta Pharmaceuticals, Inc. High mannose glycans
US20200109432A1 (en) * 2014-03-26 2020-04-09 Momenta Pharmaceuticals, Inc. Analysis of Disulfide Bonds
US20220056109A1 (en) * 2012-07-26 2022-02-24 Janssen Biotech, Inc. Glycoproteins with anti-inflammatory properties
US11352415B2 (en) 2013-05-13 2022-06-07 Momenta Pharmaceuticals, Inc. Methods for the treatment of neurodegeneration
US11661456B2 (en) 2013-10-16 2023-05-30 Momenta Pharmaceuticals, Inc. Sialylated glycoproteins
US12247071B2 (en) 2016-12-21 2025-03-11 Amgen Inc. Anti-TNF alpha antibody formulations

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2722673B1 (fr) 2012-10-17 2017-07-12 Hexal AG Procédé amélioré de mise en correspondance de glycanes de glycoprotéines
US9217168B2 (en) 2013-03-14 2015-12-22 Momenta Pharmaceuticals, Inc. Methods of cell culture
US9677105B2 (en) * 2013-03-14 2017-06-13 Momenta Pharmaceuticals, Inc. Methods of cell culture
US8956830B2 (en) 2013-03-14 2015-02-17 Momenta Pharmaceuticals, Inc. Methods of cell culture
US10450361B2 (en) 2013-03-15 2019-10-22 Momenta Pharmaceuticals, Inc. Methods related to CTLA4-Fc fusion proteins
CN106153660B (zh) * 2015-04-27 2019-05-31 上海凯赛生物技术研发中心有限公司 聚酯酰胺的鉴定方法
GB201511419D0 (en) * 2015-06-30 2015-08-12 Ge Healthcare Uk Ltd The use of bioinformatic data in autolobous cell therapies
CN114252469B (zh) * 2021-12-09 2023-12-08 江南大学 一种同步测定细菌多糖中多糖和磷含量的定量核磁方法

Citations (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859449A (en) 1987-09-14 1989-08-22 Center For Molecular Medicine And Immunology Modified antibodies for enhanced hepatocyte clearance
US5047335A (en) 1988-12-21 1991-09-10 The Regents Of The University Of Calif. Process for controlling intracellular glycosylation of proteins
US5068190A (en) 1988-09-21 1991-11-26 Noda Institute For Scientific Research N-acetylhexosamine-dehydrogenase, process for producing same, method for the quantitative analysis of N-acetylglucosamine or N-acetylgalactosamine using same and kit for use in the quantitative analysis
US5234905A (en) 1991-02-22 1993-08-10 University Of Colorado Foundation, Inc. Soluble CD4 molecules modified to prolong circulating half-life
US5340453A (en) 1989-09-27 1994-08-23 Astroscan, Ltd. Analysis of carbohydrates
US5360817A (en) 1990-04-24 1994-11-01 Biota Scientific Management Pty Ltd Derivatives and analogues of 2-deoxy-2,3-didehydro-N-acetyl neuraminic acid and their use as antiviral agents
US5370872A (en) 1991-08-12 1994-12-06 Swiss Serum And Vaccine Institute Berne Escherichia coliO-polysaccharide-protein conjugate vaccine
US5411942A (en) 1989-12-07 1995-05-02 Carlbiotech Ltd. A/S Peptide derivative, pharmaceutical preparation containing it and method for treatment of glaucoma
US5456909A (en) 1992-08-07 1995-10-10 T Cell Sciences, Inc. Glycoform fractions of recombinant soluble complement receptor 1 (sCR1) having extended half-lives in vivo
US5459031A (en) 1993-11-05 1995-10-17 Amgen Inc. Methods for controlling sialic acid derivatives in recombinant glycoproteins
US5500342A (en) 1993-08-23 1996-03-19 Takara Shuzo Co., Ltd. Method for determining sugar chain structure
US5510261A (en) 1991-11-21 1996-04-23 The Board Of Trustees Of The Leland Stanford Juniot University Method of controlling the degradation of glycoprotein oligosaccharides produced by cultured Chinese hamster ovary cells
US5554730A (en) 1993-03-09 1996-09-10 Middlesex Sciences, Inc. Method and kit for making a polysaccharide-protein conjugate
US5559103A (en) 1993-07-21 1996-09-24 Cytel Corporation Bivalent sialyl X saccharides
US5567684A (en) 1994-09-14 1996-10-22 The Regents Of The University Of California Synthetic ganglioside derivatives
US5663355A (en) 1985-11-19 1997-09-02 Cornell Research Foundation, Inc. Monosaccharide analog-based glycosidase inhbitors
US5667984A (en) 1991-05-07 1997-09-16 Oxford Glycosystems Ltd. Sequencing of oligosaccharides
US5679321A (en) 1993-06-17 1997-10-21 Glycomed Incorporated Sialic acid/fucose based medicaments
US5712254A (en) 1993-12-24 1998-01-27 Mitsubishi Chemical Corporation Sialic acid derivatives
US5723583A (en) 1990-11-23 1998-03-03 The General Hospital Corporation Antibody containing sialyl lewis X determinants
US5753454A (en) 1995-09-12 1998-05-19 Iowa State University Research Foundation, Inc. Sequencing of oligosaccharides: the reagent array-electrochemical detection method
US5759823A (en) 1991-03-18 1998-06-02 Scripps Research Institute Ogligosaccharide enzyme substrates and inhibitors: methods and compostions
US5856143A (en) 1993-05-14 1999-01-05 Bioflexin Ab N-containing saccharides and method for the synthesis of N-containing saccharides from amino-deoxy-disaccharides and amino-deoxy-oligosaccharides
US5879912A (en) 1993-07-15 1999-03-09 Neose Technologies, Inc. Method of synthesizing saccharide compositions
US5945322A (en) 1994-09-26 1999-08-31 The Rockefeller University Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them
US6030815A (en) 1995-04-11 2000-02-29 Neose Technologies, Inc. Enzymatic synthesis of oligosaccharides
US6048707A (en) 1991-08-30 2000-04-11 Glyko, Inc. Fluorophore assisted derivatization analysis of carbohydrates
US6096555A (en) 1995-07-26 2000-08-01 Dade Behring Marburg Gmbh Process for characterizing the glycosylation of glyco-proteins and for the in vitro determination of the bio-availability of glyco-proteins
US6132994A (en) 1996-07-23 2000-10-17 Seikagaku Corporation Lactosamine oligosaccharides and method for producing the same
WO2000065070A2 (fr) 1999-04-26 2000-11-02 Genentech, Inc. Procede de culture cellulaire
US6156547A (en) 1990-04-16 2000-12-05 Neose Pharmaceuticals, Inc. Apparatus for the synthesis of saccharide compositions
US6159954A (en) 1994-12-01 2000-12-12 Seikagaku Corporation Keratan sulfate oligosaccharide fraction and pharmaceutical containing the same
US6190522B1 (en) 1998-04-24 2001-02-20 Board Of Regents, The University Of Texas System Analysis of carbohydrates derivatized with visible dye by high-resolution polyacrylamide gel electrophoresis
US6218149B1 (en) 1988-09-15 2001-04-17 The Trustees Of Columbus University In The City Of New York Antibodies having modified carbohydrate content and methods of preparation and use
US6261805B1 (en) 1999-07-15 2001-07-17 Boyce Thompson Institute For Plant Research, Inc. Sialyiation of N-linked glycoproteins in the baculovirus expression vector system
US6274568B1 (en) 1998-08-06 2001-08-14 Ronald L. Schnaar Compounds for altering cell surface sialic acids and methods of use therefor
US6280989B1 (en) 1999-06-17 2001-08-28 Dmitri Kapitonov Sialyltransferases
US6284516B1 (en) 1995-01-24 2001-09-04 Shin-Etsu Bio, Inc. DNA segments and methods for increasing polysaccharide production
WO2001080884A1 (fr) 2000-04-25 2001-11-01 Idec Pharmaceuticals Corporation Administration intrathecale de rituximab pour le traitement des lymphomes du systeme nerveux central
WO2002000879A2 (fr) 2000-06-28 2002-01-03 Glycofi, Inc. Procede de production de glycoproteines modifiees
US6358710B1 (en) 1996-06-07 2002-03-19 Neorx Corporation Humanized antibodies that bind to the antigen bound by antibody NR-LU-13
US20020054878A1 (en) 1997-07-02 2002-05-09 Genentech, Inc. Anti-IgE antibodies
WO2003025133A2 (fr) 2001-09-14 2003-03-27 Mimeon, Inc. Procedes de fabrication de glycomolecules a activite amelioree, et utilisations de ces dernieres
US6597996B1 (en) 1999-04-23 2003-07-22 Massachusetts Institute Of Technology Method for indentifying or characterizing properties of polymeric units
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20040077836A1 (en) 2001-10-10 2004-04-22 Neose Technologies, Inc. Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF
US20040210396A1 (en) 2003-03-28 2004-10-21 Solutia Inc. Methods and structure for automated active pharmaceuticals development
JP2005509403A (ja) 2001-03-27 2005-04-14 スミスクライン・ビーチャム・コーポレイション ＩｇＧにおけるグリコフォームの制御
US20060040353A1 (en) 2000-06-28 2006-02-23 Davidson Robert C Production of galactosylated glycoproteins in lower eukaryotes
US20060127950A1 (en) 2004-04-15 2006-06-15 Massachusetts Institute Of Technology Methods and products related to the improved analysis of carbohydrates
US20060252672A1 (en) 2005-04-05 2006-11-09 Betenbaugh Michael J Protein N-glycosylation of eukaryotic cells using dolichol-linked oligosaccharide synthesis pathway, other N-gylosylation-increasing methods, and engineered hosts expressing products with increased N-glycosylation
WO2007011041A1 (fr) 2005-07-22 2007-01-25 Kyowa Hakko Kogyo Co., Ltd. Composition d'anticorps génétiquement modifié
CN101001875A (zh) 2004-07-21 2007-07-18 格利科菲公司 主要包含man5glcnac2糖形的免疫球蛋白
WO2007087384A2 (fr) 2006-01-23 2007-08-02 Amgen Inc. Procédés de modulation de la teneur en mannose de protéines de recombinaison
CN101137757A (zh) 2005-03-07 2008-03-05 植物研究国际公司 蘑菇的糖工程
WO2008063982A2 (fr) 2006-11-13 2008-05-29 Procell Corp Épitopes de cycloprotéine à haute teneur en mannose
WO2008128230A1 (fr) 2007-04-16 2008-10-23 Momenta Pharmaceuticals, Inc. Produits de glycoprotéines de référence et procédés associés
WO2008128228A1 (fr) 2007-04-16 2008-10-23 Momenta Pharmaceuticals, Inc. Méthodes associées à la glycosylation de surface
US20080261301A1 (en) 2000-10-06 2008-10-23 Kyowa Hakko Kogyo Co., Ltd. Antibody Composition-Producing Cell
WO2008130926A2 (fr) 2007-04-16 2008-10-30 Momenta Pharmaceuticals, Inc. Caractérisation de n-glycanes à l'aide d'exoglycosidases
US20090041770A1 (en) 2004-11-12 2009-02-12 Chamberlain Aaron Keith Fc VARIANTS WITH ALTERED BINDING TO FcRn
US20090069232A1 (en) 2007-04-03 2009-03-12 Nico Luc Marc Callewaert Glycosylation of molecules
US20090104603A1 (en) 2005-07-11 2009-04-23 Glykos Finland Oy Tissue Carbohydrate Compositions and Analysis Thereof
US20090226968A1 (en) 1999-03-02 2009-09-10 Betenbaugh Michael J Engineering Intracellular Sialylation Pathways
US20090258014A1 (en) 2008-04-11 2009-10-15 John Laterra Combination of hgf inhibitor and egf inhibitor to treat cancer
US20090311732A1 (en) 2006-12-22 2009-12-17 Ares Trading S.A. Analytical method for analyzing c-terminus truncation
US20090317834A1 (en) 2006-03-08 2009-12-24 Jarmo Laine Novel cellular glycan compositions
US20100048456A1 (en) 2003-04-09 2010-02-25 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US20100081150A1 (en) 2008-09-26 2010-04-01 Eureka Therapeutics, Inc. Methods for Generating Host Cells
US20100113294A1 (en) 2007-04-16 2010-05-06 Momenta Pharmaceuticals, Inc. Defined glycoprotein products and related methods
US20100173323A1 (en) 2006-06-09 2010-07-08 University Of Maryland, Baltimore Glycosylation engineered antibody therapy
WO2010138502A2 (fr) 2009-05-26 2010-12-02 Momenta Pharmaceuticals, Inc. Production de glycoprotéines
WO2010136492A2 (fr) 2009-05-28 2010-12-02 Glaxo Group Limited Protéines de liaison à l'antigène
WO2010141855A1 (fr) 2009-06-05 2010-12-09 Momenta Pharmaceuticals, Inc. Procédés de modulation de la fucosylation de glycoprotéines
US8034906B2 (en) 2006-10-27 2011-10-11 Abbott Biotechnology Ltd. Crystalline anti-hTNFalpha antibodies
WO2011127322A1 (fr) 2010-04-07 2011-10-13 Momenta Pharmaceuticals, Inc. Glycanes à haute teneur en mannose
WO2011127325A1 (fr) 2010-04-07 2011-10-13 Momenta Pharmaceuticals, Inc. Sélection et utilisation de cellules hôte pour la production de glycoprotéines
US20110280873A1 (en) 2010-05-11 2011-11-17 Schering Corporation MCP1-Ig FUSION VARIANTS

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU783973B2 (en) 2000-02-08 2006-01-12 Genentech Inc. Improved galactosylation of recombinant glycoproteins
CL2003002461A1 (es) * 2002-11-27 2005-01-07 Dow Chemical Company Agroscien Inmunoglobulina que comprende al menos un glicano afucosilado, composicion que la contiene, secuencia nucleotidica y vector que la comprende, procedimiento para producir dicha inmunoglobulina en plantas.
EP2686671A4 (fr) 2011-03-12 2015-06-24 Momenta Pharmaceuticals Inc N-glycanes contenant de la n-acétylhexosamine dans des produits de glycoprotéines

Patent Citations (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5663355A (en) 1985-11-19 1997-09-02 Cornell Research Foundation, Inc. Monosaccharide analog-based glycosidase inhbitors
US4859449A (en) 1987-09-14 1989-08-22 Center For Molecular Medicine And Immunology Modified antibodies for enhanced hepatocyte clearance
US6218149B1 (en) 1988-09-15 2001-04-17 The Trustees Of Columbus University In The City Of New York Antibodies having modified carbohydrate content and methods of preparation and use
US5068190A (en) 1988-09-21 1991-11-26 Noda Institute For Scientific Research N-acetylhexosamine-dehydrogenase, process for producing same, method for the quantitative analysis of N-acetylglucosamine or N-acetylgalactosamine using same and kit for use in the quantitative analysis
US5047335A (en) 1988-12-21 1991-09-10 The Regents Of The University Of Calif. Process for controlling intracellular glycosylation of proteins
US5340453A (en) 1989-09-27 1994-08-23 Astroscan, Ltd. Analysis of carbohydrates
US5411942A (en) 1989-12-07 1995-05-02 Carlbiotech Ltd. A/S Peptide derivative, pharmaceutical preparation containing it and method for treatment of glaucoma
US6156547A (en) 1990-04-16 2000-12-05 Neose Pharmaceuticals, Inc. Apparatus for the synthesis of saccharide compositions
US5360817A (en) 1990-04-24 1994-11-01 Biota Scientific Management Pty Ltd Derivatives and analogues of 2-deoxy-2,3-didehydro-N-acetyl neuraminic acid and their use as antiviral agents
US5723583A (en) 1990-11-23 1998-03-03 The General Hospital Corporation Antibody containing sialyl lewis X determinants
US5234905A (en) 1991-02-22 1993-08-10 University Of Colorado Foundation, Inc. Soluble CD4 molecules modified to prolong circulating half-life
US5759823A (en) 1991-03-18 1998-06-02 Scripps Research Institute Ogligosaccharide enzyme substrates and inhibitors: methods and compostions
US5667984A (en) 1991-05-07 1997-09-16 Oxford Glycosystems Ltd. Sequencing of oligosaccharides
US5370872A (en) 1991-08-12 1994-12-06 Swiss Serum And Vaccine Institute Berne Escherichia coliO-polysaccharide-protein conjugate vaccine
US6048707A (en) 1991-08-30 2000-04-11 Glyko, Inc. Fluorophore assisted derivatization analysis of carbohydrates
US5510261A (en) 1991-11-21 1996-04-23 The Board Of Trustees Of The Leland Stanford Juniot University Method of controlling the degradation of glycoprotein oligosaccharides produced by cultured Chinese hamster ovary cells
US5456909A (en) 1992-08-07 1995-10-10 T Cell Sciences, Inc. Glycoform fractions of recombinant soluble complement receptor 1 (sCR1) having extended half-lives in vivo
US5554730A (en) 1993-03-09 1996-09-10 Middlesex Sciences, Inc. Method and kit for making a polysaccharide-protein conjugate
US5856143A (en) 1993-05-14 1999-01-05 Bioflexin Ab N-containing saccharides and method for the synthesis of N-containing saccharides from amino-deoxy-disaccharides and amino-deoxy-oligosaccharides
US5679321A (en) 1993-06-17 1997-10-21 Glycomed Incorporated Sialic acid/fucose based medicaments
US5879912A (en) 1993-07-15 1999-03-09 Neose Technologies, Inc. Method of synthesizing saccharide compositions
US5559103A (en) 1993-07-21 1996-09-24 Cytel Corporation Bivalent sialyl X saccharides
US5500342A (en) 1993-08-23 1996-03-19 Takara Shuzo Co., Ltd. Method for determining sugar chain structure
US5459031A (en) 1993-11-05 1995-10-17 Amgen Inc. Methods for controlling sialic acid derivatives in recombinant glycoproteins
US5712254A (en) 1993-12-24 1998-01-27 Mitsubishi Chemical Corporation Sialic acid derivatives
US5567684A (en) 1994-09-14 1996-10-22 The Regents Of The University Of California Synthetic ganglioside derivatives
US5945322A (en) 1994-09-26 1999-08-31 The Rockefeller University Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them
US6159954A (en) 1994-12-01 2000-12-12 Seikagaku Corporation Keratan sulfate oligosaccharide fraction and pharmaceutical containing the same
US6284516B1 (en) 1995-01-24 2001-09-04 Shin-Etsu Bio, Inc. DNA segments and methods for increasing polysaccharide production
US6030815A (en) 1995-04-11 2000-02-29 Neose Technologies, Inc. Enzymatic synthesis of oligosaccharides
US6096555A (en) 1995-07-26 2000-08-01 Dade Behring Marburg Gmbh Process for characterizing the glycosylation of glyco-proteins and for the in vitro determination of the bio-availability of glyco-proteins
US5753454A (en) 1995-09-12 1998-05-19 Iowa State University Research Foundation, Inc. Sequencing of oligosaccharides: the reagent array-electrochemical detection method
US6358710B1 (en) 1996-06-07 2002-03-19 Neorx Corporation Humanized antibodies that bind to the antigen bound by antibody NR-LU-13
US6132994A (en) 1996-07-23 2000-10-17 Seikagaku Corporation Lactosamine oligosaccharides and method for producing the same
US20020054878A1 (en) 1997-07-02 2002-05-09 Genentech, Inc. Anti-IgE antibodies
US6190522B1 (en) 1998-04-24 2001-02-20 Board Of Regents, The University Of Texas System Analysis of carbohydrates derivatized with visible dye by high-resolution polyacrylamide gel electrophoresis
US6274568B1 (en) 1998-08-06 2001-08-14 Ronald L. Schnaar Compounds for altering cell surface sialic acids and methods of use therefor
US20090226968A1 (en) 1999-03-02 2009-09-10 Betenbaugh Michael J Engineering Intracellular Sialylation Pathways
US6597996B1 (en) 1999-04-23 2003-07-22 Massachusetts Institute Of Technology Method for indentifying or characterizing properties of polymeric units
WO2000065070A2 (fr) 1999-04-26 2000-11-02 Genentech, Inc. Procede de culture cellulaire
US6280989B1 (en) 1999-06-17 2001-08-28 Dmitri Kapitonov Sialyltransferases
US6261805B1 (en) 1999-07-15 2001-07-17 Boyce Thompson Institute For Plant Research, Inc. Sialyiation of N-linked glycoproteins in the baculovirus expression vector system
WO2001080884A1 (fr) 2000-04-25 2001-11-01 Idec Pharmaceuticals Corporation Administration intrathecale de rituximab pour le traitement des lymphomes du systeme nerveux central
WO2002000879A2 (fr) 2000-06-28 2002-01-03 Glycofi, Inc. Procede de production de glycoproteines modifiees
US20060040353A1 (en) 2000-06-28 2006-02-23 Davidson Robert C Production of galactosylated glycoproteins in lower eukaryotes
US20080261301A1 (en) 2000-10-06 2008-10-23 Kyowa Hakko Kogyo Co., Ltd. Antibody Composition-Producing Cell
JP2005509403A (ja) 2001-03-27 2005-04-14 スミスクライン・ビーチャム・コーポレイション ＩｇＧにおけるグリコフォームの制御
WO2003025133A2 (fr) 2001-09-14 2003-03-27 Mimeon, Inc. Procedes de fabrication de glycomolecules a activite amelioree, et utilisations de ces dernieres
US20090203550A1 (en) 2001-09-14 2009-08-13 Momenta Pharmaceuticals, Inc. Methods of making glycomolecules with enhanced activities and uses thereof
US20040077836A1 (en) 2001-10-10 2004-04-22 Neose Technologies, Inc. Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20040210396A1 (en) 2003-03-28 2004-10-21 Solutia Inc. Methods and structure for automated active pharmaceuticals development
US20100048456A1 (en) 2003-04-09 2010-02-25 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US20100144553A1 (en) 2004-04-15 2010-06-10 Massachusetts Institute Of Technology Methods and products related to the improved analysis of carbohydrates
US20060127950A1 (en) 2004-04-15 2006-06-15 Massachusetts Institute Of Technology Methods and products related to the improved analysis of carbohydrates
CN101001875A (zh) 2004-07-21 2007-07-18 格利科菲公司 主要包含man5glcnac2糖形的免疫球蛋白
US20090041770A1 (en) 2004-11-12 2009-02-12 Chamberlain Aaron Keith Fc VARIANTS WITH ALTERED BINDING TO FcRn
CN101137757A (zh) 2005-03-07 2008-03-05 植物研究国际公司 蘑菇的糖工程
US20060252672A1 (en) 2005-04-05 2006-11-09 Betenbaugh Michael J Protein N-glycosylation of eukaryotic cells using dolichol-linked oligosaccharide synthesis pathway, other N-gylosylation-increasing methods, and engineered hosts expressing products with increased N-glycosylation
US20090104603A1 (en) 2005-07-11 2009-04-23 Glykos Finland Oy Tissue Carbohydrate Compositions and Analysis Thereof
WO2007011041A1 (fr) 2005-07-22 2007-01-25 Kyowa Hakko Kogyo Co., Ltd. Composition d'anticorps génétiquement modifié
WO2007087384A2 (fr) 2006-01-23 2007-08-02 Amgen Inc. Procédés de modulation de la teneur en mannose de protéines de recombinaison
US20090317834A1 (en) 2006-03-08 2009-12-24 Jarmo Laine Novel cellular glycan compositions
US20100173323A1 (en) 2006-06-09 2010-07-08 University Of Maryland, Baltimore Glycosylation engineered antibody therapy
US8034906B2 (en) 2006-10-27 2011-10-11 Abbott Biotechnology Ltd. Crystalline anti-hTNFalpha antibodies
WO2008063982A2 (fr) 2006-11-13 2008-05-29 Procell Corp Épitopes de cycloprotéine à haute teneur en mannose
US20090311732A1 (en) 2006-12-22 2009-12-17 Ares Trading S.A. Analytical method for analyzing c-terminus truncation
US20090069232A1 (en) 2007-04-03 2009-03-12 Nico Luc Marc Callewaert Glycosylation of molecules
US20100129843A1 (en) 2007-04-16 2010-05-27 Momenta Pharmaceuticals, Inc. Characterization of n-glycans using exoglycosidases
US20100113294A1 (en) 2007-04-16 2010-05-06 Momenta Pharmaceuticals, Inc. Defined glycoprotein products and related methods
WO2008128230A1 (fr) 2007-04-16 2008-10-23 Momenta Pharmaceuticals, Inc. Produits de glycoprotéines de référence et procédés associés
WO2008128228A1 (fr) 2007-04-16 2008-10-23 Momenta Pharmaceuticals, Inc. Méthodes associées à la glycosylation de surface
WO2008130926A2 (fr) 2007-04-16 2008-10-30 Momenta Pharmaceuticals, Inc. Caractérisation de n-glycanes à l'aide d'exoglycosidases
US20090258014A1 (en) 2008-04-11 2009-10-15 John Laterra Combination of hgf inhibitor and egf inhibitor to treat cancer
US20100081150A1 (en) 2008-09-26 2010-04-01 Eureka Therapeutics, Inc. Methods for Generating Host Cells
WO2010138502A2 (fr) 2009-05-26 2010-12-02 Momenta Pharmaceuticals, Inc. Production de glycoprotéines
WO2010136492A2 (fr) 2009-05-28 2010-12-02 Glaxo Group Limited Protéines de liaison à l'antigène
WO2010141855A1 (fr) 2009-06-05 2010-12-09 Momenta Pharmaceuticals, Inc. Procédés de modulation de la fucosylation de glycoprotéines
WO2011127322A1 (fr) 2010-04-07 2011-10-13 Momenta Pharmaceuticals, Inc. Glycanes à haute teneur en mannose
WO2011127325A1 (fr) 2010-04-07 2011-10-13 Momenta Pharmaceuticals, Inc. Sélection et utilisation de cellules hôte pour la production de glycoprotéines
US20110280873A1 (en) 2010-05-11 2011-11-17 Schering Corporation MCP1-Ig FUSION VARIANTS

Non-Patent Citations (165)

* Cited by examiner, † Cited by third party
Title
Akiyama et al., "Analysis of the role of glycosylation of the human fibronectin receptor", J. Biol. Chem. vol. 264(30) pp. 18011-18018 (1989).
Andersen et al., "Multiple cell culture factors can affect the glycosylation of ASN-184 in CHO-produced tissue-type plasminogen activator", Biotechnol. Bioeng., 2000, vol. 70, pp. 25-31.
Andrade et al., "Solid-phase oligosaccharide synthesis: preparation of comlex structures using a novel linker and different glycosylating agents", Org. Lett., 1999, vol. 1, No. 11, pp. 1811-1814.
Anulula et al., "Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates", Analytical Biochemistry, 305(1), pp. 1-23 (2006).
Baker et al.(Biotechnology Engineering, vol. 73, No. 3, pp. 188-202). *
Baker et al., "Metabolic control of recombinant protein N-glycan processing in NSO and CHO cells", Biotechnol. Bioeng., 2001, vol. 73, pp. 188-202.
Becker et al., "Fucose: biosynthesis and biological function in mammels" Glycobiology, Jul. 13(7) pp. 41R-53R (2003).
Bohne et al., "Sweet- WWW-based rapid 3D construction of oligo- and polysaccharides", Bioinformatics, Sep. 1999, vol. 15, No. 9, pp. 767-768, XP 001024942 ISSN: 1367-4803, Oxford University Press, Surrey, GB.
Bollati-Foglin et al., "Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality", Biotechnol. Prog., 2005, vol. 21, pp. 17-21.
Bowman et al., "Biosynthesis of L-selectin ligands: sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases", Biochemistry, 2001, vol. 40, No. 18, pp. 5382-5391.
Breidenbach et al., "Targeted metabolic labeling of yeast N-glycans with unnatural sugars" Proc. Natl. Acad. Sci., vol. 107(9) pp. 3988-3993 (2010).
Broschat et al., "Purification and characterization of GDP-D-mannose 4,6-dehydratase from porcine", Thyroid. Eur. J. Biochem., 1985, vol. 153, No. 2, pp. 397-401.
C.E. Joosten et al: "Effect of Culture Conditions on the Degree of Sialylation of a Recombinant Glycoprotein Expressed in Insect Cells", Biotechnology Progress, vol. 19, No. 3, 6 pp. 739-749 (2003).
Cabrera et al., "Influence of culture conditions of the N-glycosylation of a monoclonal antibody specific for recombinant hepatitis B surface antigen", Biotechnol. Appl. Biochem., 2005, vol. 41, pp. 67-76.
Chen et al., "Independent Lec1A CHO Glycosylation Mutants Arise from Point Mutations in N-Acetylglucosaminyltransferase I that Reduce Affinity for Both Substrates. Molecular Consequences Based on the Crystal Structure of GlcNac-TI", Biochemistry vol. 40(30) pp. 8765-8772 (2001).
Chen et al., "T cell receptors signaling co-regulates multiple Golgi genes to enhance N-glycan branching" J. Boil. Chem. vol. 284(47) pp. 32454-32461 (2009).
Chen P et al: "Effects of elevated ammonium on glycosylation gene expression in CHO cells", Metabolic Engineering, Academic Press, US, vol. 8, No. 2, pp. 123-132 (2006).
Clark et al., "Gene-expression profiles for five key glycosylation genes for galactose-fed CHO cells expression recombinant IL-4/13 cytokine trap", Biotechnol. and Bioeng., 2005, vol. 90, No. 5, pp. 568-577.
Cooper et al., "GlycoSuiteDB: a curated relational database of glycoprotein glycan structures and their biological sources. 2003 update", Nucleic Acids Research, 2003, vol. 31, No. 1, pp. 511-513.
Cooper et al., "GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources", Nucleic Acids Research, 2001, vol. 29, No. 1, pp. 332-335.
Cornil et al. Tumor cell surface beta 1-4-linked galactose binds to lectin(s) on microvascular endothelial cells and contributes to organ colonization. J Cell Bioi. Aug. 1990;111 (2):773-81.
Cox et al: "Glycan Optimization of a Human Monoclonal Antibody in the Aquatic Plant Lemna Minor", Nature Biotechnology, Nature Publishing Group, New York, NY, US, vol. 24, No. 12, pp. 1591-1597 (2006).
Crowell et al., "Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system", Biotechnol. and Bioeng., 2006, pp. 538-549.
Debray et al, "Glycoprotein Analysis: General Methods", In: "Encyclodpedia of Analytical Chemistry" pp. 1-39.
Donaldson et al., "The use of lectins to select subpopulations of insect cells", Biotechnol. and Bioeng., 1999, vol. 61, pp. 616-619.
Dorka et al., "Modelliong Batch and Fed-Batch Mammalian Cell Cultures for Optimizing MAb Productivity", M.S. Thesis pp. 1-197 (2007).
Drecktrah et al., "Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tuble formation and retrograde trafficing" Mol. Biol. Cell, vol. 14(8) pp. 3459-3469 (2003).
European Patent Office, Communication pursuant to Article 96(2) mailed Oct. 30, 2007 in related European Patent Application No. 02 773 390.6.
Extended European Search Report dated Mar. 1, 2013.
Extended European Search Report from European Application No. 11766759.2 dated Aug. 19, 2013.
Extended European Search Report from European application serial No. 11766762.6 dated Jan. 28, 2014.
Extended European Search Report from European Patent Application No. 12757887.0 dated May 28, 2015.
Fareed, "S-9-10 synthetic and biotechnology derived glycomimetics", Impact on Drug Development, 2000, Database Google 6th Annual Pg Forum, Abstract.
FDA. Scientific Considerations in Demonstrating Biosimilarity to a Reference Product [online] Feb. 2012 [retrieved Dec. 10, 2013]. Available on the internet: <URL: <http://www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsAreDevelopedandApproved/d/ApprovalApplications/TheraputicBiologicApplicatios/Biosimilars>). Especially p. 1 para 2, p. 5 para 2, p. 6 para 2, p. 9 para 1-4.
Ferrara et al., "Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of golgi enzyme localization domain and co-expression of heterologous b 1, 4-N-acetylglucosaminyltransferase III and golgi a-mannosidase II", Biotechnol. and Bioeng., 2006, vol. 93, No. 5, pp. 851-861.
Fitz et al., "Combined use of subtilisin and N-acetyl neuraminic acid aldolase for the synthesis of a fluorescent sialic acid", J. Org. Chem., 1994, vol. 59, pp. 8279.
Fleischer eta l., "Mechanism of Glycosylation ion the Golgi Apparatus" The Journal of Histochemistry and Cytochemistry, vol. 31, No. 8, pp. 1033-1040 (1983).
Forno et al., "N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocytemacrophage colony-stimulating factor secreted by a Chinese hamster overy cell line", Eur. J. Biochem., 2004, vol. 271, pp. 907-919.
Freeze et al., "Use of Glycosidases to Study protein trafficking" Curr Protoc Cell Bio. (15.2.1-15.2.26) (1999).
Fukuda et al., "Survival of recombinant erythropoietin in the circulation: the role of carbohydrates", Blood, 1989, vol. 73, pp. 84-89.
Gates et al., "Glycoprotein analysis manual" internet citation, pp. 1-89, retrieved from the Internet:URL:download.bioon.com.cn/view/upload/201301/27194411-2997.pdf.
Gawlitzek et al., "Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms", Biotechnol. and Bioeng., 2000, vol. 68, No. 6, pp. 637-646.
Gawlitzek et al., "Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions", Journal of Biotechnology, 1995, vol. 42, pp. 117-131.
Goldman et al., "Monitoring recombinant human interferon-g N-glycosylation and during perfused fluidized-bed and stirred-tank batch culture of CHO cells", Biotechnol. and Bioeng., 1998, vol. 60, pp. 596-607.
Gu et al., "Improvement of interferon-g sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine", Biotechnol. and Bioeng., 1998, vol. 58, pp. 642-648.
Hara et al., "Determination of Mono-O-acetylated N-Acetylneuraminic Acids in Human and Rat Sera by Fluorometric High-Performance Liquid Chromatography" Analytical Biochemistry, 179 pp. 162-166 (1989).
Harue Imai-Nishiya et al., "Double knockdown of a 1,6 fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC", BMC Biotechnology, 2007, vol. 7, No. 84, pp. 1-13.
Hendrick V et al: "Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis", Cytotechnology, Kluwer Academic Publishers, DO, vol. 36, No. 1-3, pp. 71-83 (2001).
Hewitt et al., "Solution and solid-support synthesis of a potential leishmaniasis carbohydrate vaccine", J. Org. Chem., 2001, vol. 15, No. 66(12), pp. 4233-4243.
Hills et al., "Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NSO cells", Biotechnol. and Bioeng., 2001, vol. 75, pp. 239-251.
Hirabayashi et al., "Separation technologies for glycomics", J. Chromatog. B Analyst. Biomed. Life Sci., May 2002, vol. 771, No. 1-2, pp. 67-87, Database Medline, US National Library of Medicine, Abstract.
Hodoniczky, et al., "Control of Recombinant Monoclonal Antibody Effector Functions by Fc N-Glycan Remodeling in Vitro" Biotechnol. Prog., 21:1644-1652 (2005).
Hoja-Lukowicz et al., "High-mannose-type oligosaccharides form human placental arylsulfatase A are core fucosylated as confirmed bu MALDI MS", Gyclobiology, vol. 10, No. 6, pp. 551-557 (2000).
Hosoi S et al: "Modulation of Oligosaccharide Structure of a Pro-Urokinase Derivative (Pro-UKDeltaGS1) by Changing Culture Conditions of a Lymphoblastoid Cell Line Namalwa KJM-1 Adapted to Serum-Free Medium", Cytotechnology, Kluwer Academic Publishers, Dordrecht, NL, vol. 19, No. 2, pp. 125-135 (1996).
Hossler et al., "Systems analysis of N-glycan processing in mammalian cells" PLoS One, vol. 2(8)e713 pp. 1-17 (2007).
International Preliminary Report on Patenability including the Written Opinion from International Application Serial No. PCT/US2010/036058 mailed Nov. 19, 2010.
International Preliminary Report on Patentability and Written Opinion for International application No. PCT/US2010/037454 issued Dec. 6, 2011.
International Preliminary Report on Patentability and Written Opinion from International Application Serial No. PCT/US2008/060365 mailed Apr. 2, 2009.
International Preliminary Report on Patentability for International Application No. PCT/US2011/031637 mailed Oct. 18, 2012.
International Preliminary Report on Patentability for International Application Serial No. PCT/US2012/028759 issued Jan. 14, 2014.
International Preliminary Report on Patentability from PCT Application Serial No. PCT/US2008/060354 mailed Apr. 2, 2009.
International Preliminary Report on Patentability including the Written Opinion for International Application Serial No. PCT/US2011/031641 mailed Aug. 17, 2011.
International Search Report and Written Opinion for corresponding International Application Serial No. PCT/US2011/31637 mailed Aug. 30, 2011.
International Search Report and Written Opinion for corresponding International Application Serial No. PCT/US2013/043670 mailed Jan. 7, 2014.
International Search Report and Written Opinion from International Serial No. PCT/US13/43696 mailed Jan. 17, 2014.
International Search Report dated Jan. 7, 2014 in PCT/US2013/43671.
International Search Report for PCT/US04/04423, mailed Dec. 28, 2004.
International Search Report for PCT/US2002/29285, filing date Dec. 23, 2002.
International Search Report for PCT/US2010/36058, dated Nov. 19, 2010.
International Search Report for PCT/US2010/37454, dated Sep. 1, 2010.
International Search Report including the Written Opinion for International Application Serial No. PCT/US2011/031641 mailed Aug. 17, 2011.
International Search Report including the Written Opinion for International Application Serial No. PCT/US2012/18759 mailed Sep. 4, 2012.
International Search Report including the Written Opinion for International Application Serial No. PCT/US2013/043667 mailed Jan. 13, 2014.
International Search Report including Written Opinion for International Application Serial No. PCT/US13/43676 mailed Jan. 16, 2014.
International Search Report including Written Opinion for International Application Serial No. PCT/US13/43693 mailed Jan. 13, 2014.
International Search Report including Written Opinion for PCT/US13/43671 mailed Jan. 7, 2014.
International Search Report including Written Opinion for PCT/US2012/28759 mailed Sep. 4, 2012.
International Search Report including Written Opinion for PCT/US2013/43674 mailed Jan. 15, 2014.
International Search Report including Written Opinion for PCT/US2013/43675 mailed Dec. 23, 2013.
Jabs et al. Fast and Extensive Mass Spectrometry Characterization of Theraputic mABs: The Panitumumab Case Study [online] CASSS Mass Spec Meeting Sep. 14, 2012 Poster 125 [ retrieved Dec. 10, 2013] Available on the internet: <URL: http://archief.fhi.nl/het2012/images/6.piere-fabre.pdf.
Jefferis, "Glycosylatin of Human IgG Antibodies: Relevance to Therapeutic Applications" Biopharm. Advanstar Communications, Inc., 14(9):19-27 (2001).
Jong Hyun Nam et al: "The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells", Biotechnology and Bioengineering, vol. 100, No. 6, 4, pp. 1178-1192 (2008).
Kakehi et al., "Analysis of glycoproteins, glycopeptides and glycoprotein-derived polysaccharides by high-performance capillary electrophoresis", J. Chromatogr. A., 1996, vol. 720, No. 1-2, pp. 377-393.
Kanda et al., "Comparison of biological activity among nonfucosylated therapeutic IgF1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybride, and complex types" Glycobiology, vol. 17(1) pp. 104-118 (2007).
Kanda et al., "Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: A new strategy for generating completely non-cucosylated recombinant therapeutics" Journal of Biotechnology, vol. 130 pp. 300-310 (2007).
Kawashima et al., "Tyrosine kinase activity of epidermal growth factor receptor is regulated by GM3 binding through carbohydrate to carbohydrate interactions" J. Biol. Chem. vol. 284(10) pp. 6147-6155 (2009).
Keiser et al., "Direct isolation and sequencing of specific protein-binding glycosaminoglycans", Nature Medicine, 2001, vol. 7, No. 1, pp. 123-128.
Keppler et al., "Biosynthetic modulation of sialic acid-dependent virus-receptor interactions of two primate polyoma viruses", J. Biol. Chem., 1995, vol. 270, No. 3, pp. 1308-1314.
Kim et al., "Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophilia S2 cells", Biotechnol. and Bioeng., 2005, vol. 92, No. 4, pp. 452-461.
Kosa et al., "Modification of cell surfaces by enzymetic introduction of special sialic acid analogues", Biochm. Biophys. Res. Commun., 1993, vol. 190, pp. 914.
Krapp et al., "Structural Analysis of Human IgG-Fc Glycoforms Reveals a Correlation Between Glycosylation and Structural Integrity", Journal of Molecular Biology, 325(5) pp. 979-989 (2003).
Kunkel et al., "Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors", Biotechnol. Prog., 2000, vol. 16, pp. 462-470.
Kunkel et al., "Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody", Journal of Biotechnology, 1998, vol. 62, pp. 55-71.
Le Floch et al., "HPCE monitoring of the N-glycosylation pattern and sialylation of murine erythropoietin produced by CHO cells in batch processes", Biotechnol. Prog., 2004, vol. 20, pp. 864-871.
Lifely M R et al: "Glycosylation and biological-activity of CAMPATH-1H expressed in different cell-lines and grown under different culture conditions", Glycobiology, Oxford University Press, US, vol. 5, No. 8, pp. 813-822 (1995).
Lin et al., "Unusual stereoselectivity in sialic acid aldolase-catalyzed aldol condensations: synthesis of both enantiomers of high-carbon monosaccharides", J. Am. Chem. Soc., 1992, vol. 114, pp. 10138-10145.
Lipscomb et al., "Effect of production method and gene amplification on the glycosylation pattern of a secreted reporter protein in CHO cells", Biotechnol. Prog., 2005, vol. 21, pp. 40-49.
Live et al., "Conformational influences of a glycosylation of a peptide: a possible model for the effect of glycsylation on the rate of protein folding", Proceedings of the National Academy of Sciences of the United States of America, 1996, vol. 93, No. 23, pp. 12759-12761, XP002293395 ISSN: 0027-8424.
Lopez-Avalos et al., "The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a riole in uridine nucleotide sugar transport into Golgi vesicles", Glycobiology, vol. 11(5) pp. 413-422 (2001).
Lucocq et al. (The Journal of Histochemistry and Cytochemistry, vol. 35, No. 1, pp. 67-74, 1987). *
MacMillan et al.,"Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin", Chemistry and Biology, 2001, vol. 8, pp. 133-145.
Millward et al., "Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice" Biologicals, 36, pp. 41-47 (2008).
Moran et al., "A systematic approach to the validation of process control parameters for monoclonal antibody production in fed-batch culture of a murine myeloma", Biotechnol. and Bioeng., 2000, vol. 69, No. 3, pp. 242-255.
Mueller et al., " Recombinant glycoprotein product quality in proliferation-controlled BHK-21 cells", Biotechnol. and Bioeng., 1999, vol. 65, No. 5, pp. 529-536.
Nairn et al., "Regulation of glycan structures in animal tissues: transcript profiling of glycan-related genes" J. Biol. Chem., vol. 282(25) pp. 17298-17313 (2008).
Nyberg et al ., "Metabolic effects on recombinant interferon-g glycosylation in continuous culture of Chinese hamster ovary cells", Biotechnol. and Bioeng., 1999, vol. 62, No. 3.
Oh et al.,"Effect of N-acetylcystein on butyrate-treated Chinese hamster overy cells to improve the production of recombinant human interferon-b-1a", Biotechnol. Prog., 2005, vol. 21, pp. 1154-1164.
Pace et al., "Characterization of Minor N-linked Glycans on Antibodies Using Endo H Release and MALDI-Mass Spectrometry" Analytical Letters, vol. 42, No. 11, pp. 1711-1724 (2009).
Park et al., "Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media", Journal of Biotechnology, 2000, vol. 81, pp. 129-140.
Plante et al., "Automated solid-phase synthesis of oligosaccharides", Science, 2001, vol. 291, No. 5508, pp. 1523-1527.
Plante et al., "Formation of b-glucosamine and b-mannose linkages using glycosyl phosphates", Org. Lett., 2000, vol. 2, No. 24, pp. 3841-3843.
Reitman et al., "Mouse Lymphoma Cell Lines Resistant to Pea Letin are defective in Fucose Metabolism", J Biol Chem., vol. 255(20) pp. 9900-9906 (1980).
Restelli et al., "The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells", Biotechnology and Bioengineering, 2006, vol. 94, No. 3, pp. 481-494.
Ripka et al., "Two Chinese Hamster Ovary Glycosylation Mutants Affected in the Converstion of GOP-Mannose to GOP-Fucose" Arch Biochem Biophys vol. 249(2) pp. 533-545 (1986).
Ritzenthaler et al., "Reevaluation of the effets of brefeldin A on plant cells using tobacco Bright Yellow 2 cells expressing Golgi-targeted green fluoresent protein and COPI antisera" Plant Cell, vol. 14(1) pp. 237-261 (2002).
Robinson D K et al: "Characterization of a recombinant antibody produced in the course of a high yield fed-batch process", Biotechnology and Bioengineering, Wiley & Sons, Hoboken, NJ, US, vol. 44, No. 6, 5, pp. 727-735 (1994).
Rodriguez J et al: "Enhanced production of monomeric interferon-[beta] by CHO cells through the control of culture conditions", Biotechnology Progress, American Institute of Chemical Engineers, US, vol. 21, No. 1, pp. 22-30 (2005).
Santell et al., "Aberrant metabolic sialylation of recombinant proteins expressed in Chinese hamster ovary cells in high productivity cultures", Biochemical and Biophysical Research Communications, 1999, vol. 258, pp. 132-137.
Sasaki et al.,"Site-specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides of peptides at each glycosylation site by fast atom bombardment mass spectrometry", Biochemistry, 1988, vol. 27, pp. 8618-8626.
Schulz et al., "Mediators of galactose sensitivity in UDP=galactoe 4′-epimerase-impaired mammalian cells" J. Biol Chem, 280 (14) pp. 13493-13502 (2005).
Schuster et al., "Improved effector functions of a therapeutic monoclonal Lewis V-specific antibody by glycoform engineering", Cancer Res., 2005, vol. 65, No. 17, pp. 7934-7941.
Search Report from Chinese Application No. 201180022319.9 dated Sep. 30, 2102.
Senger et al., "Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein", Biotechnol. Prog., 2003, vol. 19, pp. 1199-1209.
Serrato et al., "Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal anitbody in hybridoma cultures", Biotechnology and Bioengineering, 2004, vol. 88, No. 2, pp. 176-188.
Shames et al., "CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives", Glycobiology, 1991, vol. 1, pp. 187-191.
Sherman, MD, RE, Biosimilar Biological Products. Biosimilar Guidance Webinar. US Food and Drug Administration pp. 1-22 (2012).
Shinkawa eta l., The absense of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem., vol. 278(5) pp. 3466-3473 (2003).
Sokolowski et al., "Conformational analysis of biantennary glycans and molecular moldeling of their complexes with lentil lectin", Journal of Molecular Graphics and Modeling, Feb. 1997, vol. 15, No. 1, pp. 37-42, 54, XP002293396 ISSN: 1093-3263.
Sparks et al., "Synthesis of potential inhibitors of hemagglutination by Influenza virus: chemoenzymic preparation of N-5 analogs of N-acetylneuraminic acid", Tetrahedron, 1993, vol. 49, pp. 1.
Spearman et al., "Production and glycosylation of recombinant â-interferon in suspension and cytopore microcarrier cultures of CHO cells", Biotechnol. Prog., 2005, vol. 21, pp. 31-39.
Srinivas et al., "Assessment of Dose Proportionality, Absolute Bioavailability, and Immunogenicity Response of CTLA4Ig (BMS-188667), a Novel Immunosuppressive Agent, Following Subcutaneous and Intravenous Administration to Rats" Pharmaceutical Research, 14(7) pp. 911-916 (1997).
Srinivas et al., "Pharmacokinetics and Pharmacodynamics of CTLA4Ig (BMS-188667), a Novel Immunosuppressive Agent, in Monkeys following Multiple Doses" Journal of Pharmaceutical Sciences, 85(1) pp. 1-4 (1996).
Sung et al., "Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells", Journal of Biotechnology, 2004, vol. 112, pp. 232-335.
Supplemental Partial European Search Report, dated Aug. 31, 2004 for Application No. 02773390.6.
Supplemental Partial European Search Report, dated Jan. 23, 2015 for Application No. EP 12757887.0.
Takeuchi et al.,"Structures and functional roles of the sugar chains of human erythropoietins", Glycobiology, 1991, vol. 1, No. 4, pp. 337-346.
Tran et al., "Separation of carbohydrate-mediated microheterogeneity of recombinant human erythropoietin by free solution capillary electrophoresis", Journal of Chromatography, 1991, vol. 542, pp. 459-471.
Trombetta et al., "Glycoprotein reglucosylation and nucleotide sugar utlization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum" The EMBO Journal, vol. 18, No. 12, pp. 3282-3292 (1999).
Trummer et al., "Process parameter shifting: part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors", Biotechnol. and Bioeng., 2006, vol. 94, No. 6, pp. 1033-1044.
Umaña et al., "Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity", Nature Biotechnology, 1999, vol. 17, pp. 176-180.
Van Berkel et al., "N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG", Biotechnology Progress, Vo;. 25, No. 1, pp. 244-251 (2009).
Van De Nieuwenhof et al., "Recombinant glycodelin carring the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cells but not in Chinese hamster ovary cells" Eur. J. Biochem, vol. 267 pp. 4753-4762 (2000).
Varki, "Radioactive tracer techniques in the sequencing of glycoprotein oligosaccharides", J. FASEB, 1991, vol. 2, pp. 226-235.
Venkataraman et al., "Sequencing complex polysaccharides", Science, 1999, vol. 286, pp. 537-542.
Von Der Lieth, "Expanding proteomics to glycobiology: biocomputing approaches understanding the function of sugar", Pacific Symposium on Biocomputing, 2002, Abstract.
Von Horsten, et al., "Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase" Glycobiology, 20(12):1607-1618 (2010).
Wang et al., "EDEM an ER quality control receptor" Nat. Struct. Biol., vol. 10(5) pp. 319-321 (2003).
Watson et al.,"Capillary electrophoresis separation of human recombinant erythropoietin (r-HuEPO) glycoforms", Analytical Biochemistry, 1993, vol. 210, pp. 389-393.
Watson et al.,"Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster overy cells", Glycobiology, 1994, vol. 4, No. 2, pp. 227-237.
Webb J W et al., Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry Analytical Biochemistry, vol. 169, pp. 337-349 (1998).
Weiner et al., "A senstive enzyme immunoassay for the quantitation of human CTLA4Ig fusion proten in mouse serum: pharmacokinetic application to optimizing cell line selection" Journal of Pharmaceutical and Biomedical Analysis, 15(5) pp. 571-579 (1997).
Wong et al., "Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures", Biotechnol. and Bioeng., 2005, vol. 89, No. 2, pp. 164-177.
Wopereis et al., "Mechanisms in Protein O-Glycan Biosynthesis and Clinical and Molecular Aspects of Protein O-Glycan Biosynthesis Defects: A Review" Clinical Chem., vol. 52(4) pp. 547-600 (2006).
Wright et al., "In vivo trafficking and catabolism of IgG1 antibodies with Fc associated carbohydrates of differing structure", Glycobiology, 2000, vol. 10, No. 12, pp. 1347-1355.
Yang et al. "Bio-Basis Function Neural Network for Prediction of Protease Cleavage Sites in Proteins" IEEE Transactions on Neural Netwroks, vol. 16, pp. 263-274 (2005).
Yang et al., "Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process", Biotechnol. and Bioeng., 2000, vol. 69, No. 1, pp. 74-82.
Yang et al., "Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture", Biotechnol. Prog., 2000, vol. 16, pp. 751-759.
Ye et al., "N-glycan branching requirement in neuronal and postnatal viability", Glycobiology, vol. 14(6) pp. 547-558 (2004).
Yoko-o et al. (Glycobiology, vol. 13, No. 8, pp. 581-589, 2003). *
Yoon et al., "Effect of culture pH on erythropoietin production by Chinese hamster overy cells grown in suspension at 32.5 and 37 degree Celsius", Biotechnol. and Bioeng., 2005, vol. 89, No. 3, pp. 345-356.
Yoon et al., "Effect of simultaneous application of stressful culture conditions on specific productivity and heterogeneity of erythropoietin in Chinese hamster overy cells", Biotechnol. Prog., 2004, vol. 20, pp. 1293-1296.
Yuen et al., "Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures", British Journal of Haematology, 2003, vol. 121, pp. 511-526.
Yuk et al., "Changes in the overall extent of protein glycosylation by Chinese hamster overy cells over the course of batch culture", Biotechnol. Appl. Biochem., 2002, vol. 36, pp. 133-140.
Yuk et al., "Glycosylation by Chinese hamster overy cells in dolichol phosphate-supplemented cultures", Biotechnol. Appl. Biochem., 2002, vol. 36, pp. 141-147.
Zhang et al., "Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-gamma from Chinese hamster ovary cell culture by hydrophilic interaction chromatography" Journal of Chromatography B: Biomedical Sciences & Applications, vol. 712, No. 1-2, pp. 73-82 (1998).

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9921210B2 (en) 2010-04-07 2018-03-20 Momenta Pharmaceuticals, Inc. High mannose glycans
US20160060675A1 (en) * 2011-03-12 2016-03-03 Momenta Pharmaceuticals, Inc. N-acetylhexosamine-containing n-glycans in glycoprotein products
US9890410B2 (en) * 2011-03-12 2018-02-13 Momenta Pharmaceuticals, Inc. N-acetylhexosamine-containing N-glycans in glycoprotein products
US20220056109A1 (en) * 2012-07-26 2022-02-24 Janssen Biotech, Inc. Glycoproteins with anti-inflammatory properties
US12559542B2 (en) * 2012-07-26 2026-02-24 Momenta Pharmaceuticals, Inc. Glycoproteins with anti-inflammatory properties
US11352415B2 (en) 2013-05-13 2022-06-07 Momenta Pharmaceuticals, Inc. Methods for the treatment of neurodegeneration
US12297256B2 (en) 2013-05-13 2025-05-13 Momenta Pharmaceuticals, Inc. Methods for the treatment of neurodegeneration
US11661456B2 (en) 2013-10-16 2023-05-30 Momenta Pharmaceuticals, Inc. Sialylated glycoproteins
US20200109432A1 (en) * 2014-03-26 2020-04-09 Momenta Pharmaceuticals, Inc. Analysis of Disulfide Bonds
US12247071B2 (en) 2016-12-21 2025-03-11 Amgen Inc. Anti-TNF alpha antibody formulations

Also Published As

Publication number Publication date
EP2686671A2 (fr) 2014-01-22
US20120295273A1 (en) 2012-11-22
WO2012125553A2 (fr) 2012-09-20
US9890410B2 (en) 2018-02-13
WO2012125553A3 (fr) 2014-02-27
EP2686671A4 (fr) 2015-06-24
CN103782168B (zh) 2016-03-16
US20160060675A1 (en) 2016-03-03
CN103782168A (zh) 2014-05-07

Similar Documents

Publication Publication Date Title
US9890410B2 (en) N-acetylhexosamine-containing N-glycans in glycoprotein products
CN102292640B (zh) 在来源于CHO细胞的糖蛋白产物中的含有半乳糖-α-1,3-半乳糖的N-聚糖
US9103821B2 (en) Methods related to modified glycans
US20110136682A1 (en) Antennary fucosylation in glycoproteins from cho cells
CA2682744C (fr) Methodes associees a la glycosylation de surface
US9029081B2 (en) Characterization of N-glycans using exoglycosidases
EP2358760B1 (fr) Caractérisation de glycanes à liaison o

Legal Events

Date Code Title Description
AS Assignment

Owner name: MOMENTA PHARMACEUTICALS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WASHBURN, NATHANIEL;AREVALO, ENRIQUE;MILLEA, KEVIN;AND OTHERS;SIGNING DATES FROM 20120530 TO 20120703;REEL/FRAME:028731/0098

STCF Information on status: patent grant

Free format text: PATENTED CASE

FEPP Fee payment procedure

Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1551); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Year of fee payment: 4

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1552); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Year of fee payment: 8